Cyclin dependent kinase inhibitors and methods of use

ABSTRACT

The presently disclosed subject matter relates to methods and compositions for protecting healthy cells from damage due to DNA damaging agents. In particular, the presently disclosed subject matter relates to the protective action of selective cyclin dependent kinase 4/6 (CDK4/6) inhibitors administered to subjects that have been exposed to or that are at risk of exposure to DNA damage.

RELATED APPLICATIONS

The presently disclosed subject matter is based on and claims thebenefit of U.S. Provisional Application Ser. No. 61/177,724, filed May13, 2009; the disclosure of which is incorporated herein by reference inits entirety.

GOVERNMENT INTEREST

The presently disclosed subject matter was made with U.S. Governmentsupport under Grant No. 2R01AG024379-06 awarded by the NationalInstitutes of Health through the National Institute on Aging. Thus, theU.S. Government has certain rights in the presently disclosed subjectmatter.

TECHNICAL FIELD

The presently disclosed subject matter relates to methods andcompositions for protecting healthy cells from DNA damage and augmentingthe efficacy of toxicity reducing agents, such as growth factors. Inaddition, the presently disclosed subject matter relates to methods andcompositions for treating autoimmune diseases by blocking theproliferation of certain immune cells. In particular, the presentlydisclosed subject matter relates to uses of selective cyclin dependentkinase 4/6 (CDK4/6) inhibitors to induce pharmacologic quiescence incertain stem and progenitor cell populations within a mammalian subjectand thereby enhancing clinical outcomes for that subject.

ABBREVIATIONS

-   -   %=percentage    -   μg=microgram    -   μL=microliters    -   μM=micromolar    -   2BrIC=2-bromo-12,13-dihydro-5H-indolo[2,3-a]pyrrolo[3,4]-carbazole-5,6-dione    -   BM=bone marrow    -   BM-MNC=bone marrow mononuclear cells    -   BrdU=5-bromo-2-deoxyuridine    -   BUN=blood area nitrogen    -   CAFC=cobblestone area-forming cell    -   CBC=complete blood count    -   CDK=cyclin-dependent kinase    -   CDK4/6=cyclin dependent kinase 4 and/or cyclin-dependent kinase        6    -   CLP=common lymphoid progenitors    -   CMP=common myeloid progenitors    -   CNS=central nervous system    -   DMEM=Dulbecco's Modified Eagle Medium    -   DMSO=dimethyl sulfoxide    -   DNA=deoxyribonucleic acid    -   DOX=doxorubicin    -   EPO=erythropoietin    -   Etop=etoposide    -   FACS=fluorescence-activated cell sorting    -   FBS=fetal bovine serum    -   g=gram    -   GC=germinal center    -   G-CSF=granulocyte colony-stimulating factor    -   GEMM=genetically engineered murine model    -   GM-CSF=granulocyte-macrophage colony stimulating factor    -   GMP=granulocyte-monocyte progenitors    -   Gy=gray    -   h=hours    -   HPLC=high performance liquid chromatography    -   HSC=hematopoietic stem cells    -   HSPC=hematopoietic stem and progenitor cells    -   IC₅₀=50% inhibitory concentration    -   IHC=immunohistochemistry    -   IL=interleukin    -   IP=intraperitoneal    -   IR=ionizing radiation    -   ITP=idiopathic thrombocytopenic purpura    -   kg=kilogram    -   LT-HSC=long term hematopoietic stem cell    -   MEP=megakaryocyte-erythroid progenitors    -   mg=milligrams    -   MPP=multipotent progenitor    -   nM=nanomolar    -   NP-CGG=nitrophenylacetyl-chicken gamma globulin    -   PD=6-acetyl-8-cyclopentyl-5-methyl-2-(5-piperazin-1-yl-pyridin-2-ylamino)-8H-pyrido-[2,3-d]-pyrimidin-7-one        (also referred to as PD 0332991)    -   RA=rheumatoid arthritis    -   RB=retinoblastoma tumor suppressor protein    -   RLU=relative light units    -   SEM=standard error of the mean    -   SLE=systemic lupus erythematosus    -   ST-HSC=short term hematopoietic stem cell    -   Sv=sievert    -   tHDF=telomerized human diploid fibroblast    -   TTP=thrombotic thrombocytopenic purpura

BACKGROUND

The treatment of cancer often includes the use of DNA damaging drugsand/or other DNA damaging agents, such as ionizing radiation. Thesetreatments can be non-specific and, particularly at high doses, toxic tonormal, rapidly dividing cells. This often leads to various side effectsin patients undergoing cancer treatment.

For example, bone marrow suppression, a severe reduction of blood cellproduction in bone marrow, is one such side effect. It is characterizedby both myelosuppression (anemia, neutropenia, agranulocytosis andthrombocytopenia) and lymphopenia. Neutropenia is characterized by aselective decrease in the number of circulating neutrophils and anenhanced susceptibility to bacterial infections. Anemia, a reduction inthe number of red blood cells or erythrocytes, the quantity ofhemoglobin, or the volume of packed red blood cells (characterized by adetermination of the hematocrit) affects approximately 67% of cancerpatients undergoing chemotherapy in the United States. See BioWorldToday, page 4, Jul. 23, 2002. Thrombcytopenia is a reduction in plateletnumber with increased susceptibility to bleeding. Lymphopenia is acommon side-effect of chemotherapy characterized by reductions in thenumbers of circulating lymphocytes (also called T- and B-cells).Lymphopenic patients are predisposed to a number of types of infections.

Thus, the medical practioner typically has to balance the efficacy ofchemotherapeutic and radiotherapeutic techniques in destroying abnormalproliferative cells with associated cytotoxic effects on normal cells.Because of this, the therapeutic index of chemotherapy and radiotherapytechniques is narrowed, often resulting in incomplete tumor reduction,tumor recurrence, increasing tumor burden, and induction of chemotherapyand/or radiation resistant tumors.

Numerous methods have been designed in an effort to reduce normal tissuedamage while still delivering effective therapeutic doses of DNAdamaging agents. With regard to IR, these techniques includebrachytherapy, fractionated and hyperfractionated dosing, complicateddose scheduling and delivery systems, and high voltage therapy with alinear accelerator. However, such techniques only attempt to strike abalance between the therapeutic and undesirable effects of theradiation, and full efficacy has not been achieved.

Small molecules have been used to reduce some of the side effects ofcertain chemotherapeutic compounds. For example, leukovorin has beenused to mitigate the effects of methotrexate on bone marrow cells and ongastrointestinal mucosa cells. Amifostine has been used to reduce theincidence of neutropenia-related fever and mucositis in patientsreceiving alkylating or platinum-containing chemotherapeutics. Also,dexrazoxane has been used to provide cardioprotection from anthracyclineanti-cancer compounds. Unfortunately, there is concern that manychemoprotectants, such as dexrazoxane and amifostine, can decrease theefficacy of chemotherapy given concomitantly.

Additional chemoprotectant therapies include the use of growth factors.Hematopoietic growth factors are available on the market as recombinantproteins. These proteins include granulocyte colony stimulating factor(G-CSF) and granulocyte-macrophage colony stimulating factor (GM-CSF)and their derivatives for the treatment of neutropenia, anderythropoietin (EPO) and its derivatives for the treatment of anemia.However, while growth factors can hasten recovery of some blood celllineages, they do not treat suppression of platelets, macrophages,T-cells or B-cells.

The non-selective kinase inhibitor staurosporine has been shown toafford protection from DNA damaging agents in some cultured cell types.See Chen et al., J. Natl. Cancer Inst., 92, 1999-2008 (2000); and Ojedaet al., Int J. Radiat. Biol., 61, 663-667 (1992). Staurosporine is anaturally occurring product and non-selective kinase inhibitor thatbinds most mammalian kinases with high affinity. See Karaman et al.,Nat. Biotechnol., 26, 127-132 (2008). Staurosporine treatment can elicitan array of cellular responses including apoptosis, cell cycle arrestand cell cycle checkpoint compromise depending on cell type, drugconcentration, and length of exposure. For example, staurosporine hasbeen shown to sensitize cells to DNA damaging agents such as ionizingradiation and chemotherapy (see Bernhard et al., Int J. Radiat. Biol.,69, 575-584 (1996); Teyssier et al., Bull. Cancer, 86, 345-357 (1999);Hallahan et al., Radiat. Res., 129, 345-350 (1992); Zhang et al., J.Neurooncol., 15, 1 -7 (1993); Guo et al., Int J. Radiat. Biol., 82,97-109 (2006); Bucher and Britten, Br. J. Cancer, 98, 523-528 (2008);Laredo et al., Blood, 84, 229-237 (1994); Luo et al., Neoplasia, 3,411-419 (2001); Wang et al., Yao Xue Xue Bao, 31, 411-415 (1996); Chenet al., J. Natl. Cancer Inst., 92, 1999-2008 (2000); and Hirose et al.,Cancer Res., 61, 5843-5849 (2001)) through several claimed mechanismsincluding abrogation of a G2 checkpoint response. The mechanism wherebystaurosporine treatment affords protection from DNA damaging agents insome cultured cell types is unclear, with a few possible mechanismssuggested including inhibition of protein kinase C or decreasing CDK4protein levels. See Chen et al., J. Natl. Cancer Inst., 92, 1999-2008(2000); and Ojeda et al., Int. J. Radiat. Biol., 61, 663-667 (1992). Noeffect of staurosporine has been shown on hematopoietic progenitors, norhas staurosporine use well after exposure to DNA damaging agents beenshown to afford protection. Further, staurosporine's non-selectivekinase inhibition has led to significant toxicities independent of itseffects on the cell cycle (e.g. hyperglycemia) after in vivoadministration to mammals and these toxicities have precluded itsclinical use.

Accordingly, there is an ongoing need for practical methods to protectsubjects who are scheduled to incur, are at risk for incurring, or whohave already incurred, exposure to DNA damaging agents and/or events andmethods of augmenting the efficacy of toxicity reducing agents. Inaddition, an ongoing need exists for methods and compositions fortreating autoimmune diseases by blocking the proliferation of immunecells.

SUMMARY

In some embodiments, the presently disclosed subject matter provides amethod of increasing the efficacy of a toxicity reducing agent in asubject in need of treatment thereof, the method comprising: providing asubject that has been exposed to, is being exposed to, or is at risk ofbeing exposed to a DNA damaging agent or event; administering to saidsubject a toxicity reducing agent; and administering to said subject apharmaceutically effective amount of a compound that selectivelyinhibits cyclin dependent kinase 4 (CDK4) and/or cyclin dependent kinase6 (CDK6).

In some embodiments, the toxicity reducing agent is a chemotherapytoxicity reducing agent. In some embodiments, the toxicity reducingagent is a radiation toxicity reducing agent.

In some embodiments, the toxicity reducing agent comprises one or moreagents selected from the group comprising, but not limited to, a growthfactor, a granulocyte colony-stimulating factor (G-CSF), a pegylatedG-CSF, granulocyte-macrophage colony stimulating factor (GM-CSF),thrombopoietin, erythropoietin, pegylated erythropoietin, interleukin(IL)-12, steel factor, a keratinocyte growth factor, or a derivativethereof.

In some embodiments, the compound that selectively inhibits CDK4 and/orCDK6 induces pharmacologic quiescence in one or more cells within thesubject. In some embodiments, the one or more cells are each selectedfrom the group comprising a hematologic cell, a hematologic stem cell,and a hematologic precursor cell.

In some embodiments, the compound that selectively inhibits CDK4 and/orCDK6 is administered to the subject prior to the subject being exposedto the DNA damaging agent or event, at the same time the subject isbeing exposed to the DNA damaging agent or event, or after exposure ofthe subject to the DNA damaging agent or event. In some embodiments, thecompound that selectively inhibits CDK4 and/or CDK6 is administered tothe subject between about 24 and about 48 hours after exposure of thesubject to the DNA damaging agent or event.

In some embodiments, the presently disclosed subject matter provides amethod of mitigating DNA damage in a non-hematologic cell or tissue in asubject in need of treatment thereof prior to or following exposure ofthe cell or tissue to a DNA damaging agent or event, the methodcomprising administering to the subject a pharmaceutically effectiveamount of a compound that selectively inhibits CDK4 and/or CDK6. In someembodiments, the non-hematologic cell or tissue is comprises a cell ortissue from one of the group comprising kidney, gut, heart, liver,brain, thyroid, skin, intestinal mucosa, auditory system, lung, bladder,ovaries, uterus, testicles, adrenals, gallbladder, pancreas, pancreaticislets, stomach, blood vessels, bone, and combinations thereof.

In some embodiments, the presently disclosed subject matter provides amethod of reducing or inhibiting memory T cell proliferation in asubject in need of treatment thereof, the method comprisingadministering to the subject a pharmaceutically effective amount of acompound that selectively inhibits CDK4 and/or CDK6 to the subject.

In some embodiments, the subject has or is at risk of developing anautoimmune or allergic disease. In some embodiments, the autoimmune orallergic disease is selected from the group comprising systemic lupuserythematosus (SLE), rheumatoid arthritis (RA), autoimmune arthritis,scleroderma, hemolytic anemia, autoimmune aplastic anemia, autoimmunegranulocytopenia, type I diabetes, thrombotic thrombocytopenic purpura(TTP), psoriasis, inflammatory bowel disease, Crohn's disease,ulcerative colitis, contact dermatitis, polymyalgia rheumatica, uveitis,immune pneumonitis, autoimmune hepatitis, immune nephritis, immuneglomerulonephritis, multiple sclerosis, autoimmune neuropathy, vitiligo,discoid lupus, Wegener's Granulomatosis, Henoch-Schoelein Purpura,sclerosing cholangitis, autoimmune thyroiditis, autoimmune myocarditis,autoimmune vasculitis, dermatomyositis, extrinsic and intrinsic reactiveairways disease (asthma), myasthenia gravis, autoimmune ovarian failure,pernicious anemia, Addison's disease, autoimmune hypoparathyroidism andother syndromes of inappropriate cellular immune response.

In some embodiments, the presently disclosed subject matter provides amethod of reducing or inhibiting B cell progenitor proliferation in asubject in need of treatment thereof, the method comprisingadministering to the subject a pharmaceutically effective amount of acompound that selectively inhibits CDK4 and/or CDK6 to the subject.

In some embodiments, the subject has or is at risk of developing anautoimmune or allergic disease. In some embodiments, the autoimmune orallergic disease is selected from the group consisting of systemic lupuserythematosus (SLE), rheumatoid arthritis (RA), scleroderma, hemolyticanemia, idiopathic thrombocytopenic purpura (ITP), acquired inhibitorsin hemophilia, thrombotic thrombocytopenic purpura (TTP), Goodpasture'ssyndrome, cold and warm agglutin diseases, cryoglobulinemia, andsyndromes of inappropriate antibody production.

In some embodiments, the presently disclosed subject matter provides amethod for mitigating an autoimmune or allergic disease in a subject inneed of treatment thereof, the method comprising administering to thesubject a pharmaceutically effective amount of a compound thatselectively inhibits CDK4 and/or CDK6, wherein said compound reduces orinhibits memory T cell proliferation, B cell progenitor proliferation,or both memory T cell proliferation and B cell progenitor proliferation.

In some embodiments, the autoimmune or allergic disease is selected fromthe group comprising systemic lupus erythematosus (SLE), rheumatoidarthritis (RA), autoimmune arthritis, scleroderma, hemolytic anemia,autoimmune aplastic anemia, autoimmune granulocytopenia, type Idiabetes, thrombotic thrombocytopenic purpura (TTP), psoriasis,inflammatory bowel disease, Crohn's disease, ulcerative colitis, contactdermatitis, polymyalgia rheumatica, uveitis, immune pneumonitis,autoimmune hepatitis, immune nephritis, immune glomerulonephritis,multiple sclerosis, autoimmune neuropathy, vitiligo, discoid lupus,Wegener's Granulomatosis, Henoch-Schoelein Purpura, sclerosingcholangitis, autoimmune thyroiditis, autoimmune myocarditis, autoimmunevasculitis, dermatomyositis, extrinsic and intrinsic reactive airwaysdisease (asthma), myasthenia gravis, autoimmune ovarian failure,pernicious anemia, Addison's disease, autoimmune hypoparathyroidismother syndromes of an inappropriate cellular immune response,Goodpasture's syndrome, cold and warm agglutin diseases,cryoglobulinemia, and syndromes of inappropriate antibody production.

In some embodiments, the presently disclosed subject matter provides amethod of treating cancer in a subject in need of treatment thereof,wherein the cancer is characterized by an increased level of cyclindependent kinase 2 (CDK2) activity or by reduced expression ofretinoblastoma tumor suppressor protein or a retinoblastoma familymember protein, the method comprising administering to the subject apharmaceutically effective amount of a compound that selectivelyinhibits CDK4 and/or CDK6.

In some embodiments, the compound that selectively inhibits CDK4 and/orCDK6 does not induce pharmacologic quiescence in cancer cells. In someembodiments, the compound that selectively inhibits CDK4 and/or CDK6increases the sensitivity of cancer cells to DNA damaging agents. Insome embodiments, the increase in sensitivity increases cancer celldeath.

In some embodiments, the increased level of CDK2 activity is associatedwith MYC protooncogene amplification or overexpression. In someembodiments, the increased level of CDK2 activity is associated withoverexpression of Cyclin E1, Cyclin E2, or Cyclin A.

In some embodiments, administration of the compound that selectivelyinhibits CDK4 and/or CDK6 mitigates hematologic toxicities associatedwith exposure to a DNA damaging agent or event. In some embodiments,administration of the compound that selectively inhibits CDK4 and/orCDK6 mitigates long-term toxicities such as secondary malignancy andmyelodysplasia associated with exposure to a DNA damaging agent orevent.

In some embodiments, the compound that selectively inhibits CDK4 and/orCDK6 is administered to the subject prior to the subject being exposedto the DNA damaging agent or event, at the same time the subject isbeing exposed to the DNA damaging agent or event, or after exposure ofthe subject to the DNA damaging agent or event. In some embodiments, thecompound that selectively inhibits CDK4 and/or CDK6 is administered tothe subject between about 24 and about 48 hours after exposure of thesubject to the DNA damaging agent or event.

In some embodiments, the presently disclosed subject matter provides amethod of mitigating chemotherapy-induced or radiotherapy-inducedsecondary malignancies of hematological or non-hematological origin in asubject, the method comprising administering to the subject apharmacologically effective amount of a compound that selectivelyinhibits CDK4 and/or CDK6. In some embodiments, the compound thatselectively inhibits CDK4 and/or CDK6 is administered to the subjectprior to or during the same time period that the subject is undergoingchemotherapy or radiation-based therapy to treat a primary malignancy.

It is an object of the presently disclosed subject matter to providemethods of protecting healthy cells in subjects from the effects of DNAdamaging agents and of treating certain conditions by administering tothe subject an effective amount of a selective CDK4/6 inhibitorcompound.

An object of the presently disclosed subject matter having been statedhereinabove, and which is achieved in whole or in part by the presentlydisclosed subject matter, other objects will become evident as thedescription proceeds when taken in connection with the accompanyingdrawings as best described hereinbelow.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: CDK4/6 inhibition potentiates the efficacy oferythropoietin-mediated recovery of the erythroid cell lineage followingDNA damage. Cohorts (8 mice per cohort) of irradiated (6.5 Gy) wild typemice (FVB/n) are given placebo, erythropoietin (EPO), a CDK4/6 inhibitor(PD0332991), or a combination of CDK4/6 inhibitor and EPO(PD0332991+EPO). Serial blood draws are performed at day 17 aftertreatment and complete blood counts assessed to determine the number ofred blood cells, various leukocytes subpopulations, and platelets. Theeffect of treatment on platelets is shown in the panel on the upperleft, red blood cells (RBC) in the panel on the upper right, hemoglobin(Hb) in the panel on the lower left, and hematocrit (HCT) in the panelon the lower right. Error bars represent+/−SEM.

FIG. 2A: CDK4/6 inhibition induces a G₁ arrest in primary human renalproximal tubule epithelial cells. Representative histograms of cellcycle analysis of Primary human renal proximal tubule epithelial cellstreated with varying concentrations of PD0332991 for 16 hours. Cellswere harvested, fixed, stained, and analyzed by flow cytometry. Data wasfitted using Mod-Fit™ software from Verity (Verity Software House,Topsham, Me., United States of America). Increasing concentrations ofCDK4/6 inhibitor produce a “clean” G1-arrest without evidence ofcytotoxicity.

FIG. 2B: CDK4/6 inhibition induces a G₁ arrest in primary human renalproximal tubule epithelial cells. Cell cycle analysis of Primary humanrenal proximal tubule epithelial cells treated with varyingconcentrations of PD0332991 for 16 hours. Cells were harvested, fixed,stained, and analyzed by flow cytometry. Data was fitted using Mod-Fit™software from Verity (Verity Software House, Topsham, Me., United Statesof America). Corresponding % of cells in G1 (diamonds), G2/M (squares)and S (triangles) are shown on the graph.

FIG. 3: CDK4/6 inhibition blocks proliferation of primary human renalproximal tubule epithelial cells. Cells were treated with varyingconcentrations of PD0332991 for 72 hours. Following incubation, cellproliferation was quantified using CellTiter-Glo® (Promega, Madison,Wis., United States of America). Data represent the mean of fourreplicates (relative light units, RLU)+/−standard deviation.

FIG. 4: CDK4/6 inhibition abrogates etoposide-induced DNA damage inprimary human renal proximal tubule epithelial cells. Cells werepretreated for 16 hours with PD0332991 followed by 8 hours withetoposide (Etop). Cells were collected, fixed and stained withanti-γH2AX FITC and analyzed by flow cytometry. Data was analyzed usingFlowJo (Treestar, Inc., Ashland, Oreg., United States of America). The %γH2AX positive cells are shown in the accompanying graph.

FIG. 5: CDK4/6 inhibition protects primary human renal proximal tubuleepithelial cells from etoposide-induced cell death. PD0332991 inhibitschemotherapy-induced cytotoxicity in a cdk4/6 dependent manner. Primaryhuman renal proximal tubule epithelial cells were incubated with 30 nMor 100 nM PD0332991 for 16 hours. Etoposide (Etop, 2.5 μM) was added for8 hours. Following incubation, the media was replaced with fresh mediaand the cells were incubated for an additional 7 days. On day 7, cellproliferation was assessed using CellTiter-Glo® (Promega, Madison, Wis.,United States of America). Error bars show+/−standard deviation.

FIG. 6: CDK4/6 inhibition blocks EdU incorporation into whole kidney inmice treated with cisplatin. Mice were treated with PD0332991 (150mg/kg) by oral gavage one hour prior to intraperitoneal (IP) injectionof cisplatin (10 mg/kg). EdU (100 μg/mouse) was given IP every 24 hoursprior to sacrifice. Kidneys were collected and single cell isolates wereprepared and stained for EdU incorporation. Proliferation was assessedby flow cytometry. Data represents % of EdU staining in untreated,cisplatin-treated and cisplatin/PD0332991 treated cells.

FIG. 7: CDK4/6 inhibition protects kidney function in mice treated withcisplatin. Cohorts of mice were treated with cisplatin (10 mg/kg IP)alone (squares), PD0332991 (150 mg/kg PO) alone (diamonds) orimmediately prior to cisplatin (10 mg/kg) (triangles). Kidney functionwas measured at day 7 by quantification of blood urea nitrogen (BUN) inmg/dL and serum creatinine (Serum Cr) in mg/dL. Data represents the meanof 6 animals per cohort+/−standard error of the mean.

FIG. 8: CDK4/6 inhibition potentiates proliferation of Rb deficientcells. Small cell lung cancer cell lines which are RB null (H69, H82,H209, H345) or with intact RB (H417) were incubated with PD0332991 for24 hours. Following incubation, media was replaced and the cells grownfor 7 days. Cell proliferation was assessed using WST-1 reagent. Eachdata point represents the mean of four replicates+/−SEM. CDK4/6inhibition increases cell proliferation of RB-deficient cell lines.

FIG. 9: CDK4/6 inhibition potentiates the efficacy of chemotherapy inmouse model of RB-deficient breast cancer. Mice were treated every 7days for three weeks with PD0332991 (150 mg/kg PO) alone, carboplatin(90 mg/kg IP) alone or in combination with the PD0332991(carboplatin+PD0332991). Data are % change in tumor volume andrepresents the mean of at least 15 animals per cohort+/−SEM.

FIG. 10: CDK4/6 inhibition potentiates the efficacy of chemotherapy inmouse model of RB-deficient breast cancer. Mice were treated every 7days for three weeks with carboplatin (90 mg/kg IP) alone (darkly shadedsquares) or in combination with PD0332991 (150 mg/kg PO; lightly shadedsquares). Data are % change in tumor volume and represents the mean ofat least 15 animals per cohort+/−SEM.

FIG. 11A: Acute inhibition of CDK4/6 selectively suppresses memory Tcell homeostatic proliferation in mice. Mice were treated with PD0332991(150 mg/kg by oral gavage). Proliferation of T-cells were assessed usingBrdU and flow cytometry.

FIG. 11B: Show graphs of the data shown in T-cell proliferation datashown in FIG. 11A.

FIG. 11C: Acute inhibition of CDK4/6 selectively suppresses germinalcenter formation in mice. Mice were treated with PD0332991 (150 mg/kg byoral gavage). Germinal center formation was assessed by Ki67immunohistochemistry.

FIG. 12: CDK4/6 inhibitors as human immunosuppressants. Experimentaldesign to test CDK4/6 inhibitors as human immunosuppressants.

FIG. 13: CDK4/6 inhibitors suppress T cell proliferation uponstimulation through TCR pathway in both memory and naïve compartments.Human peripheral blood T cells were purified using Automacs by CD3positive selection before being treated with CDK4/6 inhibitors andstimulated with PMA and Inomycin for 48 hrs. The proliferation of memory(CD45RA+) or naïve (CD45RA−) T cells after simulation by PMA andInomycin was measured by FACS staining of BrdU+ or Ki-67+ cells. Thepercentages of inhibition are shown, which indicates more inhibition ofmemory compartment than naïve compartment.

FIG. 14: CDK4/6 inhibitors suppress T cell proliferation uponstimulation through TCR pathway. T cells have more active proliferationupon stimulation through TCR pathway, which was abolished by CDK4/6inhibition. Similar inhibition was also observed in CD8+ compartment.Proliferation determined by BrdU or Ki67 incorporation. L4D=2BrIC, errorbars show+/−SEM.

FIG. 15: Changes of CD4 T cell composition after CDK4/6 inhibition.CDK4/6 inhibitors suppress T cell proliferation upon stimulation throughTCR pathway. Central memory (CCR7− CD45RA−), effector memory (CCR7+CD45RA+), Naïve (CCR7+ CD45RA+) and terminal differentiated T cells(CCR7− CD45RA+) were shown. The memory and terminal differentiated Tcell fractions as assessed by BrdU incorporation are reduced afterCDK4/6 inhibition. L4D=2BrIC. Similar results in CD8+ compartment.

FIG. 16: Changes of CD4 T cell composition after CDK4/6 inhibition.CDK4/6 inhibitors suppress T cell proliferation upon stimulation throughTCR pathway. Central memory (CCR7− CD45RA−), effector memory (CCR7+CD45RA+), Naïve (CCR7+ CD45RA+) and terminal differentiated T cells(CCR7− CD45RA+) were shown. The memory and terminal differentiated Tcell fractions as assessed by Ki67 staining are reduced after CDK4/6inhibition. L4D=2BrIC.

FIG. 17A: Preferential inhibition of memory T cell and TD cellproliferation in CD4+ T cells. Central memory (CCR7− CD45RA−), effectormemory (CCR7+ CD45RA+), Naïve (CCR7+ CD45RA+) and terminaldifferentiated T cells (CCR7− CD45RA+) are shown. Representative flowdot plots with indicated treatment: vehicle (DMSO), PD0332991 or 2BrIC.

FIG. 17B: Preferential inhibition of memory T cell and TD cellproliferation in CD4+ T cells. Graph showing the ratio of memory Tcells/Naïve T cells for data shown in FIG. 17A. The memory and terminaldifferentiated T cell fractions are reduced after CDK4/6 inhibition.L4D=2BrIC. Error bars show+/−SEM.

FIG. 17C: Preferential inhibition of memory T cell and TD cellproliferation in CD4+ T cells. Graph quantifies the % of T cells fordata shown in FIG. 17A. The memory and terminal differentiated T cellfractions are reduced after CDK4/6 inhibition. Error bars show+/−SEM.

FIG. 18: Preferential inhibition of memory T cell and TD cellproliferation in CD8+ T cells. Central memory (CCR7− CD45RA−), effectormemory (CCR7+ CD45RA+), Naïve (CCR7+ CD45RA+) and terminaldifferentiated T cells (CCR7− CD45RA+) were shown. The memory andterminal differentiated T cell fractions are reduced after CDK4/6inhibition. L4D=2BrIC.

FIG. 19: Preferential inhibition of memory T cell and TD cellproliferation. CDK4/6 inhibitors inhibit T cell activation through PMAand ionomycin. Purified human peripheral T cells were stimulated withPMA and ionomycin with or without CDK4/6 inhibitor treatment. Thefraction of activated T cells (CD25+) upon stimulation was measured byFACS. T cell activation was found to be decreased after CDK4/6 inhibitortreatment. L4D=2BrIC.

FIG. 20: CDK4/6 inhibitors suppress B cell proliferation afterstimulation through BCR. B cells were purified by Automacs selection ofCD19+ cells. The BrdU incorporation of purified human peripheral B cellswas determined after anti-IgM stimulation with or without CDK4/6inhibitor treatment. The fraction of proliferating B cells reduced ˜10fold after L4D inhibition. L4D=2BrIC

FIG. 21: CDK4/6 inhibition blocks T and B cell proliferation. Animalswere treated with CDK4/6 inhibitor (PD0332991, open bars) or vehicle(shaded bars) for 24 hours and euthanized. Splenocytes were isolated andstained for B and T cell markers. After gating on appropriatepopulations, Ki67 staining was performed as an indicator ofproliferation and S-phase. Error bars show+/−SEM.

FIG. 22: CDK4/6 inhibitors block B-cell proliferation. Animals weretreated with CDK4/6 inhibitor (150 mg/kg by daily oral gavage) orvehicle for 4 days, and BrdU in the drinking water for 3 days. AfterBrdU treatment, animals were euthanized. Splenocytes were isolated andstained for B cell markers. After gating on appropriate populations,BrdU staining was performed as an indicator of proliferation.

FIG. 23: CDK4/6 inhibitors block Thymopoiesis. Animals were treated withCDK4/6 inhibitor (150 mg/kg by daily oral gavage) or vehicle for 4 days,and then thymocyte number assessed by flow cytometry for Double negative(DN:CD4−CD8−), Double Positive (DP:CD4+ CD8+) or CD4 or CD8 singlypositive cells. CDK4/6 inhibition produced a pronounced decrease in theproduction of new DP cells, with modest effects on the DN and SPfractions.

DETAILED DESCRIPTION

The presently disclosed subject matter will now be described more fullyhereinafter with reference to the accompanying Examples, in whichrepresentative embodiments are shown. The presently disclosed subjectmatter can, however, be embodied in different forms and should not beconstrued as limited to the embodiments set forth herein. Rather, theseembodiments are provided so that this disclosure will be thorough andcomplete, and will fully convey the scope of the embodiments to thoseskilled in the art.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this presently described subject matter belongs. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety.

Throughout the specification and claims, a given chemical formula orname shall encompass all active optical and stereoisomers, as well asracemic mixtures where such isomers and mixtures exist.

I. DEFINITIONS

While the following terms are believed to be well understood by one ofordinary skill in the art, the following definitions are set forth tofacilitate explanation of the presently disclosed subject matter.

Following long-standing patent law convention, the terms “a”, “an”, and“the” refer to “one or more” when used in this application, includingthe claims. Thus, for example, reference to “a compound” or “a cell”includes a plurality of such compounds or cells, and so forth.

The term “comprising”, which is synonymous with “including” “containing”or “characterized by” is inclusive or open-ended and does not excludeadditional, unrecited elements or method steps. “Comprising” is a termof art used in claim language which means that the named elements areessential, but other elements can be added and still form a constructwithin the scope of the claim.

As used herein, the phrase “consisting of” excludes any element, step,or ingredient not specified in the claim. When the phrase “consists of”appears in a clause of the body of a claim, rather than immediatelyfollowing the preamble, it limits only the element set forth in thatclause; other elements are not excluded from the claim as a whole.

As used herein, the phrase “consisting essentially of” limits the scopeof a claim to the specified materials or steps, plus those that do notmaterially affect the basic and novel characteristic(s) of the claimedsubject matter.

With respect to the terms “comprising”, “consisting of”, and “consistingessentially of”, where one of these three terms is used herein, thepresently disclosed and claimed subject matter can include the use ofeither of the other two terms.

The term “and/or” when used in describing two items or conditions, e.g.,CDK4 and/or CDK6, refers to situations where both items or conditionsare present or applicable and to situations wherein only one of theitems or conditions is present or applicable. Thus, a CDK4 and/or CDK6inhibitor can be a compound that inhibits both CDK4 and CDK6, a compoundthat inhibits only CDK4, or a compound that only inhibits CDK6.

By “healthy cell” or “normal cell” is meant any cell in a subject thatdoes not display characteristics, symptoms and/or markers of a disease(such as, but not limited to, cancer or another proliferative disease).In some embodiments, the healthy cell is a stem cell. In someembodiments, the healthy cell is a hematopoietic stem or progenitor cell(HSPC). Progenitor cells include, but are not limited to, long termhematopoietic stem cells (LT-HSCs), short term hematopoietic stem cells(ST-HSCs), multipotent progenitors (MPPs), common myeloid progenitors(CMPs), common lymphoid progenitors (CLPs), granulocyte-monocyteprogenitors (GMPs), and megakaryocyte-erythroid progenitors (MEPs).Progenitor cells can also include mature effector cells derived fromhemtopoietic stem cells, including, but not limited to, erthyrocytes,platelets, granulocytes, macrophages, T-cells, and B-cells.

In some embodiments, the healthy cell is a cell in a non-hematopoetictissue, such as, but not limited to, the liver, kidney, pancreas, brain,lung, adrenals, intestine, gut, stomach, skin, auditory system, bone,bladder, ovaries, uterus, testicles, gallbladder, thyroid, heart,pancreatic islets, blood vessels, and the like.

By “DNA damaging agent or event” is meant herein both DNA damagingchemical compounds, and other effectors of DNA damage (e.g., ionizingradiation). Thus, a DNA damaging agent or event can includechemotherapeutic and radiation treatment provided for a particularpurpose, such as but not limited to a medical purpose (e.g., to treatcancer or other diseases related to overproliferation of cells). DNAdamaging agents and events can also relate to accidental exposure to DNAdamaging chemical compounds and/or other agents that can take place, forexample, due to unexpected environmental exposure (e.g., in theworkplace or in another environment due to, for example, a chemicalspill, improper disposal or other improper handling of chemical orradiological waste, failure of safety measures and/or personalprotective gear during the use of DNA damaging chemicals or radiation,terrorist attack, warfare, or industrial and/or nuclear power plantaccident).

As used herein the term “ionizing radiation” refers to radiation ofsufficient energy that, when absorbed by cells and tissues, typicallyinduces formation of reactive oxygen species and DNA damage. Ionizingradiation can include X-rays, gamma rays, and particle bombardment(e.g., neutron beam, electron beam, protons, mesons, and others), and isused for purposes including, but not limited to, medical testing andtreatment, scientific purposes, industrial testing, manufacturing, andsterilization, and weapons and weapons development. Radiation isgenerally measured in units of absorbed dose, such as the rad or gray(Gy), or in units of dose equivalence, such as rem or sievert (Sv).

By “at risk of being exposed to a DNA damaging agent or event” is meanta subject scheduled for (such as by scheduled radiotherapy orchemotherapy sessions) exposure to a DNA damaging agent or event in thefuture or a subject having a chance of being exposed to a DNA damagingagent or event inadvertently in the future. Inadvertent exposureincludes accidental or unplanned environmental or occupational exposure(e.g., terrorist attack with a radiological or chemical weapon, achemical spill or radiation leak, or exposure to a radiological orchemical weapon on the battlefield).

The term “cancer” as used herein refers to diseases caused byuncontrolled cell division and the ability of cells to metastasize, orto establish new growth in additional sites. The terms “malignancy”,“neoplasm”, “tumor” and variations thereof refer to cancerous cells orgroups of cancerous cells.

Specific types of cancer include, but are not limited to, skin cancers,connective tissue cancers, adipose cancers, breast cancers, lungcancers, stomach cancers, pancreatic cancers, ovarian cancers, cervicalcancers, uterine cancers, anogenital cancers, kidney cancers, bladdercancers, colon cancers, prostate cancers, head and neck cancers, braincancers, central nervous system (CNS) cancers, retinal cancer, blood,and lymphoid cancers.

In some embodiments, the term cancer refers to a cancer that can becharacterized by (e.g., that has cells that exhibit) an increased levelof CDK2 activity or by reduced expression of retinoblastoma tumorsuppressor protein or retinoblastoma family member protein(s), such as,but not limited to p107 and p130. The increased level of CDK2 activityor reduced expression of retinoblastoma tumor suppressor protein orretinoblastoma family member protein(s) can be increased or reduced, forexample, compared to normal cells. In some embodiments, the increasedlevel of CDK2 activity can be associated with (e.g., can result from orbe observed along with) MYC protooncogene amplification oroverexpression. In some embodiments, the increased level of CDK2activity can be associated with overexpression of Cyclin E1, Cyclin E2,or Cyclin A.

As used herein the term “chemotherapy” refers to treatment with acytotoxic compound (such as but not limited to a DNA damaging compound)to reduce or eliminate the growth or proliferation of undesirable cells,such as, but not limited to, cancer cells. Thus, as used herein,“chemotherapeutic compound” refers to a cytotoxic compound used to treatcancer. The cytotoxic effect of the compound can be, but is not requiredto be, the result of one or more of nucleic acid intercalation orbinding, DNA or RNA alkylation, inhibition of RNA or DNA synthesis, theinhibition of another nucleic acid-related activity (e.g., proteinsynthesis), or any other cytotoxic effect.

Thus, a “cytotoxic compound” can be any one or any combination ofcompounds also described as “antineoplastic” agents or “chemotherapeuticagents.” Such compounds include, but are not limited to, DNA damagingcompounds and other chemicals that can kill cells. “DNA damagingcompounds” include, but are not limited to, alkylating agents, DNAintercalators, protein synthesis inhibitors, inhibitors of DNA or RNAsynthesis, DNA base analogs, topoisomerase inhibitors, and telomeraseinhibitors or telomeric DNA binding compounds. For example, alkylatingagents include alkyl sulfonates, such as busulfan, improsulfan, andpiposulfan; aziridines, such as a benzodizepa, carboquone, meturedepa,and uredepa; ethylenimines and methylmelamines, such as altretamine,triethylenemelamine, triethylenephosphoramide,triethylenethiophosphoramide, and trimethylolmelamine; nitrogen mustardssuch as chlorambucil, chlornaphazine, cyclophosphamide, estramustine,iphosphamide, mechlorethamine, mechlorethamine oxide hydrochloride,melphalan, novembichine, phenesterine, prednimustine, trofosfamide, anduracil mustard; and nitroso ureas, such as carmustine, chlorozotocin,fotemustine, lomustine, nimustine, and ranimustine.

Antibiotics used in the treatment of cancer include dactinomycin,daunorubicin, doxorubicin, idarubicin, bleomycin sulfate, mytomycin,plicamycin, and streptozocin. Chemotherapeutic antimetabolites includemercaptopurine, thioguanine, cladribine, fludarabine phosphate,fluorouracil (5-FU), floxuridine, cytarabine, pentostatin, methotrexate,and azathioprine, acyclovir, adenine β-1-D-arabinoside, amethopterin,aminopterin, 2-aminopurine, aphidicolin, 8-azaguanine, azaserine,6-azauracil, 2′-azido-2′-deoxynucleosides, 5-bromodeoxycytidine,cytosine β-1-D-arabinoside, diazooxynorleucine, dideoxynucleosides,5-fluorodeoxycytidine, 5-fluorodeoxyuridine, and hydroxyurea.

Chemotherapeutic protein synthesis inhibitors include abrin,aurintricarboxylic acid, chloramphenicol, colicin E3, cycloheximide,diphtheria toxin, edeine A, emetine, erythromycin, ethionine, fluoride,5-fluorotryptophan, fusidic acid, guanylyl methylene diphosphonate andguanylyl imidodiphosphate, kanamycin, kasugamycin, kirromycin, andO-methyl threonine. Additional protein synthesis inhibitors includemodeccin, neomycin, norvaline, pactamycin, paromomycine, puromycin,ricin, shiga toxin, showdomycin, sparsomycin, spectinomycin,streptomycin, tetracycline, thiostrepton, and trimethoprim. Inhibitorsof DNA synthesis, include alkylating agents such as dimethyl sulfate,mitomycin C, nitrogen and sulfur mustards; intercalating agents, such asacridine dyes, actinomycins, adriamycin, anthracenes, benzopyrene,ethidium bromide, propidium diiodide-intertwining; and other agents,such as distamycin and netropsin. Topoisomerase inhibitors, such ascoumermycin, nalidixic acid, novobiocin, and oxolinic acid; inhibitorsof cell division, including colcemide, colchicine, vinblastine, andvincristine; and RNA synthesis inhibitors including actinomycin D,α-amanitine and other fungal amatoxins, cordycepin (3′-deoxyadenosine),dichlororibofuranosyl benzimidazole, rifampicine, streptovaricin, andstreptolydigin also can be used as the DNA damaging compound

Thus, current chemotherapeutic compounds whose toxic effects can bemitigated by the presently disclosed selective CDK4/6 inhibitorsinclude, but are not limited to, adrimycin, 5-fluorouracil (5FU),etoposide, camptothecin, actinomycin-D, mitomycin, cisplatin, hydrogenperoxide, carboplatin, procarbazine, mechlorethamine, cyclophosphamide,ifosfamide, melphalan, chlorambucil, bisulfan, nitrosurea, dactinomycin,daunorubicin, doxorubicin, bleomycin, plicomycin, tamoxifen, taxol,transplatinum, vinblastin, and methotrexate, and the like.

By “toxicity reducing agent” is meant a compound or other agent that isused to reduce the cytotoxic effects of an agent or event, such as butnot limited to a DNA damaging agent or event. In some embodiments, thetoxicity reducing agent is a compound that is other than a compound thatselectively inhibits one or more cyclin dependent kinase(s). Thetoxicity reducing agent is an agent that can prevent or reduce DNAdamage in a cell, tissue or subject treated with or otherwise exposed toa DNA damaging agent or event. The prevention or reducing of DNA damageeffected by the toxicity reducing agent can affect certain cells (e.g.,certain healthy) in a subject while not providing any effect in othercells (e.g., in diseased and/or tumor cells) in a subject. Thus, the useof the toxicity reducing agent can protect certain cells in a subject inorder to allow more frequent or higher dose use of DNA damaging agentsduring a disease treatment regime. In some embodiments, the toxicityreducing agent reduces undesired cytotoxicity due to the use of achemotherapeutic agent. In some embodiments, the toxicity reducing agentcan reduce undesired cytotoxicity resulting from radiation.

In some embodiments, the toxicity reducing agent is a growth factor orother naturally occurring compound, or a derivative thereof. In someembodiments, the toxicity reducing agent is selected from the groupcomprising, but not limited to a growth factor, a granulocytecolony-stimulating factor (G-CSF), a pegylated G-CSF,granulocyte-macrophage colony stimulating factor (GM-CSF),thrombopoietin, erythropoietin, pegylated erythropoietin, interleukin(IL)-12, steel factor, a keratinocyte growth factor, or to derivativesthereof (e.g., chemically modified compounds having structures basedupon one of the foregoing named toxicity reducing agents, such asalkylated or esterified derivatives).

“Increasing the efficacy of a toxicity reducing agent” refers to theability of a selective CDK4 and/or CDK6 inhibitor to increase theefficacy of a toxicity reducing agent. Thus, the term can refer tobeneficial use of a combination of a toxicity reducing agent and aselective CDK4 and/or CDK6 inhibitor. For example, use of thecombination can result in higher tolerance of the subject to a givenamount or to a given frequency of administration of a DNA damaging agentor event that the tolerance the subject would have had when given thetoxicity reducing agent (or selective CDK4 and/or CDK6 inhibitor) alone.The use of the combination can provide a higher level of protection froma side effect caused by a DNA damaging event (such as but not limited toa greater reduction in myelosuppression or a lower probability ofoccurrence of a secondary malignancy). The use of the combination canalso provide protection from a wider range of side effects due toexposure to the DNA damaging agent or event and/or protection in a widervariety of types of cells and/or tissues in the subject. For instance,in some embodiments, a selective CDK4 and/or CDK6 inhibitor can providesynergistic effects when used in combination with a growth factor torescue and support the various hematopoietic populations from a DNAdamaging agent or event.

By “pharmaceutically effect amount of a compound” is meant an amounteffective to provide a beneficial result in the subject. For example, itcan be the amount effective to reduce or eliminate the toxicityassociated with the DNA damaging agent or event (e.g., the chemotherapyor other exposure to a cytotoxic compound in healthy HSPCs in thesubject, or the IR). In some embodiments, the effective amount is theamount required to temporarily (e.g., for a few hours or days) inhibitthe proliferation of hematopoietic stem cells (i.e., to induce aquiescent state in hematopoietic stem cells) in the subject.

In some embodiments, the compound that selectively inhibits CDK4 and/orCDK6 is free of off-target effects. “Free of” can refer to a selectiveCDK4/6 inhibitor compound not having an undesired or off-target effect,particularly when used in vivo or assessed via a cell-based assay. Thus,“free of” can refer to a selective CDK4/6 inhibitor not havingoff-target effects such as, but not limited to, long term toxicity,anti-oxidant effects, estrogenic effects, tyrosine kinase inhibitoryeffects, inhibitory effects on CDKs other than CDK4/6; and/or cell cyclearrest in CDK4/6-independent cells.

A selective CDK4/6 inhibitor that is “substantially free” of off-targeteffects is a CDK4/6 inhibitor that can have some minor off-targeteffects that do not interfere with the inhibitor's ability to provideprotection from cytotoxic compounds in CDK4/6-dependent cells. Forexample, a CDK4/6 inhibitor that is “substantially free” of off-targeteffects can have some minor inhibitory effects on other CDKs (e.g.,IC₅₀s for CDK1 or CDK2 that are >0.5 μM; >1.0 μM, or >5.0 μM), so longas the inhibitor provides selective G1 arrest in CDK4/6-dependent cells.

By “reduced” or “prevented” or grammatical variations thereof means,respectively, lessening the effects or keeping the effects fromoccurring completely. “Mitigating” can refer to reducing and/orpreventing.

By “pharmacologic quiescence” is meant a temporary arrest of cellcycling.

By “at risk of developing an autoimmune disease” refers to a subjectthat is suspected of having a likelihood of developing an autoimmunedisease for reasons including, but not limited to, for example, due tohaving one or more genetic marker associated with an autoimmune disease,having a family history of autoimmune disease, and/or having hadexposure to an environmental agent that is suspected of triggering theonset of an autoimmune disease. The term can also apply to subjects thathave been diagnosed with an autoimmune disease previously but who are inremission and/or are currently symptom-free.

In some embodiments, the subject treated in the presently disclosedsubject matter is desirably a human subject, although it is to beunderstood the methods described herein are effective with respect toall vertebrate species (e.g., mammals, birds, etc.), which are intendedto be included in the term “subject.”

More particularly, provided herein is the treatment of mammals, such ashumans, as well as those mammals of importance due to being endangered(such as Siberian tigers), of economical importance (animals raised onfarms for consumption by humans) and/or social importance (animals keptas pets or in zoos) to humans, for instance, carnivores other thanhumans (such as cats and dogs), swine (pigs, hogs, and wild boars),ruminants (such as cattle, oxen, sheep, giraffes, deer, goats, bison,and camels), and horses. Thus, embodiments of the methods describedherein include the treatment of livestock, including, but not limitedto, domesticated swine (pigs and hogs), ruminants, horses, and the like.

As used herein the term “alkyl” refers to C₁₋₂₀ inclusive, linear (i.e.,“straight-chain”), branched, or cyclic, saturated or at least partiallyand in some cases fully unsaturated (i.e., alkenyl andalkynyl)hydrocarbon chains, including for example, methyl, ethyl,propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, hexyl, octyl,ethenyl, propenyl, butenyl, pentenyl, hexenyl, octenyl, butadienyl,propynyl, butynyl, pentynyl, hexynyl, heptynyl, and allenyl groups.“Branched” refers to an alkyl group in which a lower alkyl group, suchas methyl, ethyl or propyl, is attached to a linear alkyl chain. “Loweralkyl” refers to an alkyl group having 1 to about 8 carbon atoms (i.e.,a C₁₋₈ alkyl), e.g., 1, 2, 3, 4, 5, 6, 7, or 8 carbon atoms. “Higheralkyl” refers to an alkyl group having about 10 to about 20 carbonatoms, e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms.In certain embodiments, “alkyl” refers, in particular, to C₁₋₈straight-chain alkyls. In other embodiments, “alkyl” refers, inparticular, to C₁₋₈ branched-chain alkyls.

Alkyl groups can optionally be substituted (a “substituted alkyl”) withone or more alkyl group substituents, which can be the same ordifferent. The term “alkyl group substituent” includes but is notlimited to alkyl, substituted alkyl, halo, arylamino, acyl, hydroxyl,aryloxyl, alkoxyl, alkylthio, arylthio, aralkyloxyl, aralkylthio,carboxyl, alkoxycarbonyl, oxo, and cycloalkyl. There can be optionallyinserted along the alkyl chain one or more oxygen, sulfur or substitutedor unsubstituted nitrogen atoms, wherein the nitrogen substituent ishydrogen, lower alkyl (also referred to herein as “alkylaminoalkyl”), oraryl.

Thus, as used herein, the term “substituted alkyl” includes alkylgroups, as defined herein, in which one or more atoms or functionalgroups of the alkyl group are replaced with another atom or functionalgroup, including for example, alkyl, substituted alkyl, halogen, aryl,substituted aryl, alkoxyl, hydroxyl, nitro, amino, alkylamino,dialkylamino, sulfate, and mercapto.

The term “aryl” is used herein to refer to an aromatic moiety that canbe a single aromatic ring, or multiple aromatic rings that are fusedtogether, linked covalently, or linked to a common group, such as, butnot limited to, a methylene or ethylene moiety. The common linking groupalso can be a carbonyl, as in benzophenone, or oxygen, as indiphenylether, or nitrogen, as in diphenylamine. The term “aryl”specifically encompasses heterocyclic aromatic compounds. The aromaticring(s) can comprise phenyl, naphthyl, biphenyl, diphenylether,diphenylamine and benzophenone, among others. In particular embodiments,the term “aryl” means a cyclic aromatic comprising about 5 to about 10carbon atoms, e.g., 5, 6, 7, 8, 9, or 10 carbon atoms, and including 5-and 6-membered hydrocarbon and heterocyclic aromatic rings.

The aryl group can be optionally substituted (a “substituted aryl”) withone or more aryl group substituents, which can be the same or different,wherein “aryl group substituent” includes alkyl, substituted alkyl,aryl, substituted aryl, aralkyl, hydroxyl, alkoxyl, aryloxyl,aralkyloxyl, carboxyl, carbonyl, acyl, halo, nitro, alkoxycarbonyl,aryloxycarbonyl, aralkoxycarbonyl, acyloxyl, acylamino, aroylamino,carbamoyl, alkylcarbamoyl, dialkylcarbamoyl, arylthio, alkylthio,alkylene, and —NR′R″, wherein R′ and R″ can each be independentlyhydrogen, alkyl, substituted alkyl, aryl, substituted aryl, and aralkyl.

Thus, as used herein, the term “substituted aryl” includes aryl groups,as defined herein, in which one or more atoms or functional groups ofthe aryl group are replaced with another atom or functional group,including for example, alkyl, substituted alkyl, halogen, aryl,substituted aryl, alkoxyl, hydroxyl, nitro, amino, alkylamino,dialkylamino, sulfate, and mercapto.

Specific examples of aryl groups include, but are not limited to,cyclopentadienyl, phenyl, furan, thiophene, pyrrole, pyran, pyridine,imidazole, benzimidazole, isothiazole, isoxazole, pyrazole, pyrazine,triazine, pyrimidine, quinoline, isoquinoline, indole, carbazole, andthe like.

The term “heteroaryl” refers to aryl groups wherein at least one atom ofthe backbone of the aromatic ring or rings is an atom other than carbon.Thus, heteroaryl groups have one or more non-carbon atoms selected fromthe group including, but not limited to, nitrogen, oxygen, and sulfur.

As used herein, the term “acyl” refers to an organic carboxylic acidgroup wherein the —OH of the carboxyl group has been replaced withanother substituent (i.e., as represented by RCO—, wherein R is an alkylor an aryl group as defined herein). As such, the term “acyl”specifically includes arylacyl groups, such as an acetylfuran and aphenacyl group. Specific examples of acyl groups include acetyl andbenzoyl.

“Cyclic” and “cycloalkyl” refer to a non-aromatic mono- or multicyclicring system of about 3 to about 10 carbon atoms, e.g., 3, 4, 5, 6, 7, 8,9, or 10 carbon atoms. The cycloalkyl group can be optionally partiallyunsaturated. The cycloalkyl group also can be optionally substitutedwith an alkyl group substituent as defined herein, oxo, and/or alkylene.There can be optionally inserted along the cyclic alkyl chain one ormore oxygen, sulfur or substituted or unsubstituted nitrogen atoms,wherein the nitrogen substituent is hydrogen, alkyl, substituted alkyl,aryl, or substituted aryl, thus providing a heterocyclic group.Representative monocyclic cycloalkyl rings include cyclopentyl,cyclohexyl, and cycloheptyl. Multicyclic cycloalkyl rings includeadamantyl, octahydronaphthyl, decalin, camphor, camphane, andnoradamantyl.

The terms “heterocycle” or “heterocyclic” refer to cycloalkyl groups(i.e., non-aromatic, cyclic groups as described hereinabove) wherein oneor more of the backbone carbon atoms of a cyclic ring is replaced by aheteroatom (e.g., nitrogen, sulfur, or oxygen). Examples of heterocyclesinclude, but are not limited to, tetrahydrofuran, tetrahydropyran,morpholine, dioxane, piperidine, piperazine, and pyrrolidine.

“Alkoxyl” or “alkoxy” refers to an alkyl-O— group wherein alkyl is aspreviously described. The term “alkoxyl” as used herein can refer to,for example, methoxyl, ethoxyl, propoxyl, isopropoxyl, butoxyl,t-butoxyl, and pentoxyl. The term “oxyalkyl” can be used interchangablywith “alkoxyl”.

“Aryloxyl” or “aryloxy” refers to an aryl-O— group wherein the arylgroup is as previously described, including a substituted aryl. The term“aryloxyl” as used herein can refer to phenyloxyl or hexyloxyl, andalkyl, substituted alkyl, halo, or alkoxyl substituted phenyloxyl orhexyloxyl.

“Aralkyl” refers to an aryl-alkyl- group wherein aryl and alkyl are aspreviously described, and included substituted aryl and substitutedalkyl. Exemplary aralkyl groups include benzyl, phenylethyl, andnaphthylmethyl.

“Aralkyloxyl” or “aralkyloxy” refers to an aralkyl-O— group wherein thearalkyl group is as previously described. An exemplary aralkyloxyl groupis benzyloxyl.

The term “amino” refers to the —NR′R″ group, wherein R′ and R″ are eachindependently selected from the group including H and substituted andunsubstituted alkyl, cycloalkyl, heterocycle, aralkyl, aryl, andheteroaryl. In some embodiments, the amino group is —NH₂. “Aminoalkyl”and “aminoaryl” refer to the —NR′R″ group, wherein R′ is as definedhereinabove for amino and R″ is substituted or unsubstituted alkyl oraryl, respectively.

“Acylamino” refers to an acyl-NH— group wherein acyl is as previouslydescribed.

The term “carbonyl” refers to the —(C═O)— or a double bonded oxygensubstituent attached to a carbon atom of a previously named parentgroup.

The term “carboxyl” refers to the —COOH group.

The terms “halo”, “halide”, or “halogen” as used herein refer to fluoro,chloro, bromo, and iodo groups.

The terms “hydroxyl” and “hydroxy” refer to the —OH group.

The term “oxo” refers to a compound described previously herein whereina carbon atom is replaced by an oxygen atom.

The term “cyano” refers to the —CN group.

The term “nitro” refers to the —NO₂ group.

The term “thio” refers to a compound described previously herein whereina carbon or oxygen atom is replaced by a sulfur atom.

II. COMPOUNDS AND METHODS OF PROTECTION FROM DNA DAMAGING AGENTS OREVENTS

Tissue-specific stem cells and subsets of other resident proliferatingcells are capable of self-renewal, meaning that they are capable ofreplacing themselves throughout the adult mammalian lifespan throughregulated replication. Additionally, stem cells divide asymmetrically toproduce “progeny” or “progenitor” cells that in turn produce variouscomponents of a given organ. For example, in the hematopoietic system,the hematopoietic stem cells give rise to progenitor cells which in turngive rise to all the differentiated components of blood (e.g., whiteblood cells, red blood cells, lymphocytes and platelets).

The presently disclosed subject matter relates, in part, to particularbiochemical requirements of early hematopoietic stem/progenitor cells(HSPC) and other proliferating cells in the adult mammal. In particular,it has been found that certain specific proliferating cells, such asHSPC, require the enzymatic activity of the proliferative kinasescyclin-dependent kinase 4 (CDK4) and/or cyclin-dependent kinase 6 (CDK6)for cellular replication. In contrast, the vast majority ofproliferating cells in adult mammals do not require the activity of CDK4and/or CDK6 (i.e., CDK4/6). These differentiated cells can proliferatein the absence of CDK4/6 activity by using other proliferative kinases,such as cyclin-dependent kinase 2 (CDK2) or cyclin-dependent kinase 1(CDK1). Therefore, it is believed that treatment of mammals with aselective CDK4/6 inhibitor can lead to inhibition of proliferation(i.e., pharmacologic quiescence) in very restricted cellularcompartments, such as HSPC. For instance, transient treatment (such as,but not limited to, over a less than 48, 24, 20, 16, 12, 10, 8, 6, 4, 2,or 1 hour period) with PD 0332991, a selective CDK4/6 inhibitor, rendershematopoietic stem cells and their associated hematopoietic progenitorcells quiescent. Cells that are quiescent are believed to be moreresistant to the cytotoxic effects of DNA damaging agents or events thanare proliferating cells.

Accordingly, the presently disclosed subject matter provides, in someembodiments, a methods of protecting mammals from the acute and chronictoxic effects of chemotherapeutic compounds by forcing hematopoieticstem and progenitor cells (HSPCs) into a quiescent state by transient(such as, but not limited to, over a less than 48, 24, 20, 16, 12, 10,8, 6, 4, 2, or 1 hour period) treatment with an non-toxic, selectiveCDK4/6 inhibitor (such as but not limited to, an orally available,non-toxic CDK4/6 inhibitor). During the period of quiescence, thesubject's HSPC are more resistant to certain effects of thechemotherapeutic compound. The HSPCs recover from this period oftransient quiescence, and then function normally after treatment withthe inhibitor is stopped. Thus, treatment with selective CDK4/6inhibitors can provide marked bone marrow protection and can lead to amore rapid recovery of peripheral blood cell counts (hematocrit,platelets, lymphocytes, and myeloid cells) after chemotherapy and/orradiotherapy.

U.S. Pat. No. 6,369,086 to Davis et al. (hereinafter “the '086 Patent”)appears to describe that selective CDK inhibitors can be useful inlimiting the toxicity of cytotoxic agents and can be used to protectfrom chemotherapy-induced alopecia. In particular, the '086 Patentdescribes oxindole compounds as specific CDK2 inhibitors. A relatedjournal reference (see Davis et al., Science, 291, 134-137 (2001))appears to describe that the inhibition of CDK2 produces cell cyclearrest, reducing the sensitivity of the epithelium to cell cycle-activeantitumor agents and can prevent chemotherapy-induced alopecia. However,this journal reference was later retracted due to the irreproducibilityof the results. In contrast to these purported protective effects ofselective CDK2 inhibitors, for which a question is raised by theretraction of the journal article, the presently disclosed subjectmatter relates in some embodiments to protection of HSPCs and protectionfrom hematological toxicity.

The ability to protect stem/progenitor cells is desirable both in thetreatment of cancer and in mitigating the effects of accidental exposureto or overdose with cytotoxic chemicals, radiation, or other DNAdamaging agents. The protective effects of the selective CDK4/6inhibitors can be provided to the subject via pretreatment with theinhibitor (i.e., prior CDK4/6 inhibitor treatment of a subject scheduledto be treated with or at risk of exposure to a DNA damaging agent),concomitant treatment with the CDK4/6 inhibitor and the DNA damagingagent, or post-treatment with the CDK4/6 inhibitor (i.e., treatment withthe CDK4/6 inhibitor following exposure to the DNA damaging agent).Thus, in some embodiments, the presently disclosed methods relates tothe use of selective CDK4/6 inhibitory compounds to provide protectionto subjects undergoing or about to undergo treatment withchemotherapeutic compounds or radiation, and to protect subjects fromother exposure to cytotoxic compounds and/or radiation.

As used herein the term “selective CDK4/6 inhibitor compound” refers toa compound that selectively inhibits at least one of CDK4 and CDK6 orwhose predominant mode of action is through inhibition of CDK4 and/orCDK 6. Thus, selective CDK4/6 inhibitors are compounds that generallyhave a lower 50% inhibitory concentration (IC₅₀) for CDK4 and/or CDK6than for other kinases. In some embodiments, the selective CDK4/6inhibitor can have an IC₅₀ for CDK4 or CDK6 that is at least 2, 3, 4, 5,6, 7, 8, 9, or 10 times lower than the compound's IC₅₀s for other CDKs(e.g., CDK1 and CDK2). In some embodiments, the selective CDK4/6inhibitor can have an IC₅₀ for CDK4 or CDK6 that is at least 20, 30, 40,50, 60, 70, 80, 90, or 100 times lower than the compound's IC₅₀s forother CDKs. In some embodiments, the selective CDK4/6 inhibitor can havean IC₅₀ that is more than 100 times or more than 1000 times less thanthe compound's IC₅₀s for other CDKs. In some embodiments, the selectiveCDK4/6 inhibitor compound is a compound that selectively inhibits bothCDK4 and CDK6. In some embodiments, the CDK4/6 inhibitor is not anaturally occurring compound (e.g., an isoflavone). In some embodiments,the CDK4/6 inhibitor is a poor inhibitor (e.g., >1 μM in vitro IC₅₀) ofone or more tyrosine kinases. In some embodiments, the CDK4/6 inhibitoris a high potency inhibitor of serine and/or theonine kinases. In someembodiments, the CDK4/6 inhibitor is a poor CDK1 inhibitor (e.g.,(e.g., >1 μM in vitro IC₅₀). In some embodiments, the CDK4/6 inhibitoris characterized by having a 10-fold or 50-fold or 100-fold or greaterrelative potency for inhibiting CDK4 or CDK6 as compared to CDK1.

In some embodiments, the selective CDK4/6 inhibitor compound is acompound that selectively induces G1 cell cycle arrest in CDK4/6dependent cells. Thus, when treated with the selective CDK4/6 inhibitorcompound according to the presently disclosed methods, the percentage ofCDK4/6-dependent cells in the G1 phase increase, while the percentage ofCDK4/6-dependent cells in the G2/M phase and S phase decrease. In someembodiments, the selective CDK4/6 inhibitor is a compound that inducessubstantially pure (i.e., “clean”) G1 cell cycle arrest in theCDK4/6-dependent cells (e.g., wherein treatment with the selectiveCDK4/6 inhibitor induces cell cycle arrest such that the majority ofcells are arrested in G1 as defined by standard methods (e.g., propidiumiodide staining or others) and with the population of cells in the G2/Mand S phases combined being 20%, 15%, 12%, 10%, 8%, 6%, 5%, 4%, 3%, 2%,1% or less of the total cell population).

While staurosporine, a non-specific kinase inhibitor, has been reportedto indirectly induce G1 arrest in some cell types (see Chen et al., J.Nat. Cancer Inst., 92, 1999-2008 (2000)), selective CDK4/6 inhibitorscan directly and selectively induce G1 cell cycle arrest in cells, suchas specific fractions of HSPCs, to provide chemoprotection andradioprotection with reduced long term toxicity and without the need forprolonged (e.g., 48 hour or longer) treatment with the inhibitor priorto exposure with the DNA damaging agent. In particular, while somenonselective kinase inhibitors can cause G1 arrest in some cell types bydecreasing CDK4 protein levels, benefits of the presently disclosedmethods are, without being bound to any one theory, believed to be dueat least in part to the ability of selective CDK4/6 inhibitors todirectly inhibit the kinase activity of CDK4/6 in HSPCs withoutdecreasing their cellular concentration.

In some embodiments, the selective CDK4/6 inhibitor compound is acompound that is substantially free of off target effects, particularlyrelated to inhibition of kinases other than CDK4 and or CDK6. In someembodiments, the selective CDK4/6 inhibitor compound is a poor inhibitor(e.g., >1 μM IC₅₀) of CDKs other than CDK4/6 (e.g., CDK 1 and CDK2). Insome embodiments, the selective CDK4/6 inhibitor compound does notinduce cell cycle arrest in CDK4/6-independent cells. In someembodiments, the selective CDK4/6 inhibitor compound is a poor inhibitor(e.g., >1 μM IC₅₀) of tyrosine kinases. Additional, undesirableoff-target effects include, but are not limited to, long term toxicity,anti-oxidant effects, and estrogenic effects.

Anti-oxidant effects can be determined by standard assays known in theart. For example, a compound with no significant anti-oxidant effects isa compound that does not significantly scavenge free-radicals, such asoxygen radicals. The anti-oxidant effects of a compound can be comparedto a compound with known anti-oxidant activity, such as genistein. Thus,a compound with no significant anti-oxidant activity can be one that hasless than about 2, 3, 5, 10, 30, or 100 fold anti-oxidant activityrelative to genistein. Estrogenic activities can also be determined viaknown assays. For instance, a non estrogenic compound is one that doesnot significantly bind and activate the estrogen receptor. A compoundthat is substantially free of estrogenic effects can be one that hasless than about 2, 3, 5, 10, 20, or 100 fold estrogenic activityrelative to a compound with estrogenic activity, e.g., genistein.

Selective CDK4/6 inhibitors that can be used according to the presentlydisclosed methods include any known small molecule (e.g., <1000 Daltons,<750 Daltons, or less than <500 Daltons), selective CDK4/6 inhibitor, orpharmaceutically acceptable salt thereof. In some embodiments, theinhibitor is a non-naturally occurring compound (i.e., a compound notfound in nature). Several classes of chemical compounds have beenreported as having CDK4/6 inhibitory ability (e.g., in cell freeassays). Selective CDK4/6 inhibitors useful in the presently disclosedmethods can include, but are not limited to, pyrido[2,3-d]pyrimidines(e.g., pyrido[2,3-d]pyrimidin-7-ones and2-amino-6-cyano-pyrido[2,3-d]pyrimidin-4-ones), triaminopyrimidines,aryl[a]pyrrolo[3,4-d]carbazoles, nitrogen-containingheteroaryl-substituted ureas, 5-pyrimidinyl-2-aminothiazoles,benzothiadiazines, acridinethiones, and isoquinolones.

In some embodiments, the pyrido[2,3-d]pyrimidine is apyrido[2,3-d]pyrimidinone. In some embodiments thepyrido[2,3-d]pyrimidinone is pyrido[2,3-d]pyrimidin-7-one. In someembodiments, the pyrido[2,3-d]pyrimidin-7-one is substituted by anaminoaryl or aminoheteroaryl group. In some embodiments, thepyrido[2,3-d]pyrimidin-7-one is substituted by an aminopyridine group.In some embodiments, the pyrido[2,3-d]pyrimidin-7-one is a2-(2-pyridinyl)amino pyrido[2,3-d]pyrimidin-7-one. For example, thepyrido[2,3-d]pyrimidin-7-one compound can have a structure of Formula(II) as described in U.S. Patent Publication No. 2007/0179118 to Barvianet al., herein incorporated by reference in its entirety. In someembodiments, the pyrido[2,3-d]pyrimidine compound is6-acetyl-8-cyclopentyl-5-methyl-2-(5-piperazin-1-yl-pyridin-2-ylamino)-8H-pyrido-[2,3-d]pyrimidin-7-one(i.e., PD 0332991) or a pharmaceutically acceptable salt thereof. SeeToogood et al., J. Med. Chem., 2005, 48, 2388-2406.

In some embodiments, the pyrido[2,3-d]pyrimidinone is a2-amino-6-cyano-pyrido[2,3-d]pyrimidin-4-ones. Selective CDK4/6inhibitors comprising a 2-amino-6-cyano-pyrido[2,3-d]pyrimidin-4-one aredescribed, for example, by Tu et al. See Tu et al., Bioorg. Med. Chem.Lett., 2006, 16, 3578-3581.

As used herein, “triaminopyrimidines” are pyrimidine compounds whereinat least three carbons in the pyrimidine ring are substituted by groupshaving the formula —NR₁R₂, wherein R₁ and R₂ are independently selectedfrom the group consisting of H, alkyl, aralkyl, cycloalkyl, heterocycle,aryl, and heteroaryl. Each R₁ and R₂ alkyl, aralkyl, cycloalkyl,heterocycle, aryl, and heteroaryl groups can further be substituted byone or more hydroxyl, halo, amino, alkyl, aralkyl, cycloalkyl,heterocyclic, aryl, or heteroaryl groups. In some embodiments, at leastone of the amino groups is an alkylamino group having the structure—NHR, wherein R is C₁-C₆ alkyl. In some embodiments, at least one aminogroup is a cycloalkylamino group or a hydroxyl-substitutedcycloalkylamino group having the formula —NHR wherein R is C₃-C₇cycloalkyl, substituted or unsubstituted by a hydroxyl group. In someembodiments, at least one amino group is a heteroaryl-substitutedaminoalkyl group, wherein the heteroaryl group can be furthersubstituted with an aryl group substituent.

Aryl[a]pyrrolo[3,4-d]carbazoles include, but are not limited tonapthyl[a]pyrrolo[3,4-c]carbazoles, indolo[a]pyrrolo[3,4-c]carbazoles,quinolinyl[a]pyrrolo[3,4-c]carbazoles, andisoquinolinyl[a]pyrrolo[3,4-c]carbazoles. See e.g., Engler et al.,Bioorg. Med. Chem. Lett., 2003, 13, 2261-2267; Sanchez-Martinez et al.,Bioorg. Med. Chem. Lett., 2003, 13, 3835-3839; Sanchez-Martinez et al.,Bioorg. Med. Chem. Lett., 2003, 13, 3841-3846; Zhu et al., Bioorg. Med.Chem. Lett., 2003, 13, 1231-1235; and Zhu et al., J. Med. Chem., 2003,46, 2027-2030. Suitable aryl[a]pyrrolo[3,4-d]carbazoles are alsodisclosed in U.S. Patent Publication Nos. 2003/0229026 and 2004/0048915.

Nitrogen-containing heteroaryl-substituted ureas are compoundscomprising a urea moiety wherein one of the urea nitrogen atoms issubstituted by a nitrogen-containing heteraryl group.Nitrogen-containing heteroaryl groups include, but are not limited to,five to ten membered aryl groups including at least one nitrogen atom.Thus, nitrogen-containing heteroaryl groups include, for example,pyridine, pyrrole, indole, carbazole, imidazole, thiazole, isoxazole,pyrazole, isothiazole, pyrazine, triazole, tetrazole, pyrimidine,pyridazine, purine, quinoline, isoquinoline, quinoxaline, cinnoline,quinazoline, benzimidazole, phthalimide and the like. In someembodiments, the nitrogen-containing heteroaryl group can be substitutedby one or more alkyl, cycloalkyl, heterocyclic, aralkyl, aryl,heteroaryl, hydroxyl, halo, carbonyl, carboxyl, nitro, cyano, alkoxyl,or amino group. In some embodiments, the nitrogen-containing heteroarylsubstituted urea is a pyrazole-3-yl urea. The pyrazole can be furthersubstituted by a cycloalkyl or heterocyclic group. In some embodiments,the pyrazol-3-yl urea is:

See Ikuta, et al., J. Biol. Chem., 2001, 276, 27548-27554. Additionalureas that can be used according to the presently disclosed subjectmatter include the biaryl urea compounds of Formula (I) described inU.S. Patent Publication No. 2007/0027147. See also, Honma et al., J.Med. Chem., 2001, 44, 4615-4627; and Honma et al., J. Med. Chem., 2001,44, 4628-4640.

Suitable 5-pyrimidinyl-2-aminothiazole CDK4/6 inhibitors are describedby Shimamura et al. See Shimamura et al., Bioorg. Med. Chem. Lett.,2006, 16, 3751-3754. In some embodiments, the5-pyrimidinyl-2-aminothiazole has the structure:

Useful benzothiadiazine and acridinethiones compounds include those, forexample, disclosed by Kubo et al. See Kubo et al., Clin. Cancer Res.1999, 5, 4279-4286 and in U.S. Patent Publication No. 2004/0006074,herein incorporated by reference in their entirety. In some embodiments,the benzothiadiazine is substituted by one or more halo, haloaryl, oralkyl group. In some embodiments, the benzothiadiazine is selected fromthe group consisting of4-(4-fluorobenzylamino)-1,2,3-benzothiadiazine-1,1-dioxide,3-chloro-4-methyl-4H-benzo[e][1,2,4]thiadiazine-1,1-dioxide, and3-chloro-4-ethyl-4H-benzo[e][1,2,4]thiadiazine-1,1-dioxide. In someembodiments, the acridinethione is substituted by one or more amino oralkoxy group. In some embodiments, the acridinethione is selected fromthe group consisting of 3-amino-10H-acridone-9-thione (3ATA),9(10H)-acridinethione, 1,4-dimethoxy-10H-acridine-9-thione, and2,2′-diphenyldiamine-bis-[N,N′-[3-amido-N-methylamino)-10H-acridine-9-thione]].

In some embodiments, the subject of the presently disclosed methods willbe a subject who has been exposed to, is being exposed to, or isscheduled to be exposed to, a DNA damaging agent while undergoingtherapeutic treatment for a proliferative disorder. Such disordersinclude cancerous and non-cancer proliferative diseases. For example,the presently disclosed compounds are believed effective in protectinghealthy HSPCs during chemotherapeutic treatment of a broad range oftumor types, including but not limited to the following: breast,prostate, ovarian, skin, lung, colorectal, brain (i.e., glioma) andrenal.

Ideally, it is preferable that the selective CDK4/6 inhibitor notcompromise the efficacy of the DNA damaging agent by itself arrestingthe growth of the cancer cells. Most cancers appear not to depend on theactivities of CDK4/6 for proliferation as they can use the proliferativekinases promiscuously (e.g., can use CDK 1/2/4/or 6) or lack thefunction of the retinoblastoma tumor suppressor protein (RB), which isinactivated by the CDKs. Therefore, isolated inhibition of CDK4/6 shouldnot adversely affect the DNA damaging agent response in the majority ofcancers. As would be understood by one of skill in the art upon a reviewof the instant disclosure, the potential sensitivity of certain tumorsto CDK4/6 inhibition can be deduced based on tumor type and moleculargenetics. Cancers that are not expected to be affected by the inhibitionof CDK4/6 are those that can be characterized by one or more of thegroup including, but not limited to, increased activity of CDK1 or CDK2,loss or absence of retinoblastoma tumor suppressor protein (RB), highlevels of MYC expression, increased cyclin E (e.g., E1 or E2) andincreased cyclin A, or expression of a RB-inactivating protein (such asHPV-encoded E7). Such cancers can include, but are not limited to, smallcell lung cancer, retinoblastoma, HPV positive malignancies likecervical cancer and certain head and neck cancers, MYC amplified tumorssuch as Burkitts Lymphoma, and triple negative breast cancer; certainclasses of sarcoma, certain classes of non-small cell lung carcinoma,certain classes of melanoma, certain classes of pancreatic cancer,certain classes of leukemia, certain classes of lymphoma, certainclasses of brain cancer, certain classes of colon cancer, certainclasses of prostate cancer, certain classes of ovarian cancer, certainclasses of uterine cancer, certain classes of thyroid and otherendocrine tissue cancers, certain classes of salivary cancers, certainclasses of thymic carcinomas, certain classes of kidney cancers, certainclasses of bladder cancer and certain classes of testicular cancers.

For example, in some embodiments, the cancer is selected from a smallcell lung cancer, retinoblastoma and triple negative (ER/PR/Her2negative) or “basal-like” breast cancer. Small cell lung cancer andretinoblastoma almost always inactivate the retinoblastoma tumorsuppressor protein (RB), and therefore does not require CDK4/6 activityto proliferate. Thus, CDK4/6 inhibitor treatment will effectpharmacologic quiescence in the bone marrow and other normal host cells,but not in the tumor. Triple negative (basal-like) breast cancer is alsoalmost always genetically or functionally RB-null. Also, certain virallyinduced cancers (e.g. cervical cancer and subsets of Head and Neckcancer) express a viral protein (E7) which inactivates RB making thesetumors functionally RB-null. Some lung cancers are also believed to becaused by HPV. As would be understood by one of skill in the art,cancers that are not expected to be affected by CDK4/6 inhibitors (e.g.,those that are RB-null, that express viral protein E7, or thatoverexpress MYC) can be determined through methods including, but notlimited to, DNA analysis, immunostaining, Western blot analysis, andgene expression profiling.

Selective CDK4/6 inhibitors can also be used in protecting healthy HSPCsduring DNA damaging agent treatments of abnormal tissues in non-cancerproliferative diseases, including but not limited to the following:hemangiomatosis in infants, secondary progressive multiple sclerosis,chronic progressive myelodegenerative disease, neurofibromatosis,ganglioneuromatosis, keloid formation, Paget's Disease of the bone,fibrocystic disease of the breast, Peronies and Duputren's fibrosis,restenosis and cirrhosis. Further, selective CDK4/6 inhibitors can beused to ameliorate the effects of DNA damaging agents in the event ofaccidental exposure or overdose (e.g., methotrexate overdose). Thus, thepresently disclosed methods can be used to protect chemical and nuclearplant workers, scientific researchers, and emergency responders fromoccupational exposure, for example, in the event of a chemical spill orradiation leak.

According to the presently disclosed subject matter, the DNA damagingagent can be administered to a subject on any schedule and in any doseconsistent with the prescribed course of treatment, as long as theselective CDK4/6 inhibitor compound is administered prior to, during, orfollowing the administration of the DNA damaging agent. Generally,selective CDK4/6 inhibitor compound can be administered to the subjectduring the time period ranging from 24 hours prior to exposure with theDNA damaging agent until 24 hours following exposure. However, this timeperiod can be extended to time earlier that 24 hour prior to exposure tothe DNA damaging agent (e.g., based upon the time it takes the any DNAdamaging chemical compound used to achieve suitable plasmaconcentrations and/or the DNA damaging compound's plasma half-life).Further, the time period can be extended longer than 24 hours followingexposure to the DNA damaging agent so long as later administration ofthe CDK4/6 inhibitor leads to at least some protective effect. Suchpost-exposure treatment can be especially useful in cases of accidentalexposure or overdose.

In some embodiments, the selective CDK4/6 inhibitor can be administeredto the subject at a time period prior to the administration of the DNAdamaging agent, so that plasma levels of the selective CDK4/6 inhibitorare peaking at the time of administration of the DNA damaging agent. Ifconvenient, the selective CDK4/6 inhibitor can be administered at thesame time as the DNA damaging agent, in order to simplify the treatmentregimen. In some embodiments, the chemoprotectant and DNA damagingagent(s) can be provided in a single formulation.

If desired, multiple doses of the selective CDK4/6 inhibitor compoundcan be administered to the subject. Alternatively, the subject can begiven a single dose of the selective CDK4/6 inhibitor.

In some embodiments, selective CDK4/6 inhibitors can be used togetherwith other compounds or treatments to reduce undesirable effects of DNAdamaging agents or events. For example, in some embodiments, thepresently disclosed subject matter relates to methods of increasing theefficacy of a toxicity reducing agent in a subject in need of treatmentthereof, the method comprising: providing a subject that has beenexposed to, is being exposed to, or is at risk of being exposed to a DNAdamaging agent or event; administering to said subject a toxicityreducing agent; and administering to said subject a pharmaceuticallyeffective amount of a compound that selectively inhibits CDK4 and/orCDK6.

The toxicity reducing agent can be any known toxicity reducing agent.Ideally, the toxicity reducing agent is free of selective CDK4/6inhibitory activity.

In some embodiments, the toxicity reducing agent is an agent that isbeing used to or is known to have the ability to reduce undesirablecytotoxicity/side effects related to the use of (or exposure to) achemotherapeutic. In some embodiments, the toxicity reducing agent is anagent that is being used to, or is known to have the ability to, reduceundesirable toxicity/side effects related to the use of (or exposure to)radiation. Thus, in some embodiments, the toxicity reducing agent is achemoprotectant or a radioprotectant.

In some embodiments, the toxicity reducing agent is an agent being usedso that a higher dose of a chemotherapuetic or of radiation can betolerated by a subject being treated for cancer or another proliferativedisease. In some embodiments, the toxicity reducing agent is being usedso that a subject being treated for cancer of another proliferativedisease can be treated with a chemotherapeutic or radiation morefrequently. In some embodiments, the toxicity reducing agent is used toreduce or prevent side effects associated with the use of the DNAdamaging agent, such as, but not limited to, nausea, vomiting, hairloss, anemia, fatigue, peripheral neuropathy, bleeding problems,diarrhea, constipation, and the like.

In some embodiments, the toxicity reducing agent is a growth factor orother naturally occurring compound, or a derivative thereof. In someembodiments, the toxicity reducing agent is selected from the groupcomprising, but not limited to, growth factors, a granulocytecolony-stimulating factor (G-CSF), a pegylated G-CSF,granulocyte-macrophage colony stimulating factor (GM-CSF),thrombopoietin, erythropoietin (EPO), pegylated erythropoietin,interleukin (IL)-12, steel factor, a keratinocyte growth factor, or toderivatives (e.g., chemically modified compounds having structures basedupon one of the foregoing named toxicity reducing agents, such asalkylated or esterified derivatives) or combinations thereof.

In some embodiments, the use of the toxicity reducing agent and theselective CDK4/6 inhibitor can result in synergistic protective effectsfrom the DNA damaging agent or event. In some embodiments, the compoundthat selectively inhibits CDK4 and/or CDK6 induces pharmacologicquiescence in one or more cells within the subject. For example,transient treatment (e.g., over a period of about 48 hours or less) withthe compound that selectively inhibits CDK4 and/or CDK6 can temporarilyinduce pharmacologic quiescence in one or more cells within the subject.In some embodiments, the one or more cells that are induced in topharmacologic quiescence are, for example, hematologic cells,hematologic stem cells, and/or hematologic precursor cells. Thus, insome embodiments, a growth factor and a selective CDK4/6 inhibitorcompound can be used in a method to provide synergistic effects in therescue and support of various hematopoietic populations from a DNAdamaging agent or event.

In some embodiments, the selective CDK4/6 inhibitor and the toxicityreducing agent can be used in combination to rescue and support variousnon-hematologic tissues from a DNA damaging agent or event, such asionizing radiation or a chemotherapeutic. The non-hematologic tissuescan include, but are not limited to, cells or tissue from the kidney,gut, heart, liver, brain, thyroid, skin, intestinal mucosa, auditorysystem, lung, bladder, ovaries, uterus, testicles, adrenals,gallbladder, pancreas, pancreatic islets, stomach, blood vessels, bone,and combinations thereof.

The toxicity reducing agent the compound that selectively inhibits CDK4and/or CDK6 can be administered together (e.g., in the same formulationor at the same time in separate formulations) or at different times.Either or both of the toxicity reducing agent and the CDK4/6 inhibitorcan be given as a single dose or in multiple doses. In some embodiments,either the CDK4/6 inhibitor or the toxicity reducing agent can beadministered prior to the exposure to the DNA damaging agent or event,while the other of the CDK4/6 inhibitor and the toxicity reducing agentcan be administer during or after exposure to the DNA damaging agent orevent. In some embodiments, both the CDK4/6 inhibitor and the toxicityreducing agent can be administered during exposure to the DNA damagingagent (e.g., during administration of chemo or radiotherapy).Alternatively both can be administered prior to or after exposure to theDNA damaging agent. In some embodiments, the compound that selectivelyinhibits CDK4 and/or CDK6 is administered to the subject between about24 and about 48 hours (e.g., about 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46 or 48 hours)after exposure of the subject to the DNA damaging agent or event.

In some embodiments, the presently disclosed subject matter is relatedto the ability of selective CDK4/6 inhibitors to protect non-hematologiccells or tissues from DNA damaging agents or events. Thus, in someembodiments, the presently disclosed subject matter provides a method ofmitigating DNA damage in a non-hematologic cell or tissue in a subjectin need of treatment thereof prior to or following exposure of the cellor tissue to a DNA damaging agent or event, wherein the method comprisesadministering to the subject a pharmaceutically effective amount of acompound that selectively inhibits CDK4/6.

In some embodiments, the non-hematologic cell or tissue is comprises acell or tissue from the group including, but not limited to cells ortissue from the kidney, gut, heart, liver, brain, thyroid, skin,intestinal mucosa, auditory system, lung, bladder, ovaries, uterus,testicles, adrenals, gallbladder, pancreas, pancreatic islets, stomach,blood vessels, bone, and combinations thereof. In some embodiments, theDNA damaging agent is a chemotherapeutic agent, such as, but not limitedto, kanamycin, ifosfamide, camptothecin, cyclophosphamide,L-asparaginase, doxorubicin, daunorubicin, methotrexante, irinotecan,cisplatin, streptozotocin, 6-mercaptipurine, bleomycin, busulphan,vincristine, and combinations thereof. Thus, for example, the presentlydisclosed methods can relate to the use of CDK4/6 inhibitors to protectthe kidney cells from chemotherapy-induced epithelial cell damage.

Selective CDK4/6 inhibition appears to have different effects on primaryand memory immune responses. In some embodiments, the presentlydisclosed subject matter is related to the finding that selective CDK4/6inhibitors preferentially reduce memory T cell proliferation as comparedto naive T cell proliferation. Thus, in some embodiments, the presentlydisclosed subject matter provides a method of reducing or inhibitingmemory T cell proliferation in a subject in need of treatment thereof,wherein the method comprises administering to the subject apharmaceutically effective amount of a compound that selectivelyinhibits CDK4/6.

In some embodiments, the subject has or is at risk of developing anautoimmune or allergic disease, such as, but not limited to, systemiclupus erythematosus (SLE), rheumatoid arthritis (RA), autoimmunearthritis, scleroderma, hemolytic anemia, autoimmune aplastic anemia,autoimmune granulocytopenia, type I diabetes, thromboticthrombocytopenic purpura (TTP), psoriasis, inflammatory bowel disease,Crohn's disease, ulcerative colitis, contact dermatitis, polymyalgiarheumatica, uveitis, immune pneumonitis, autoimmune hepatitis, immunenephritis, immune glomerulonephritis, multiple sclerosis, autoimmuneneuropathy, vitiligo, discoid lupus, Wegener's Granulomatosis,Henoch-Schoelein Purpura, sclerosing chloangitis, autoimmunethyroiditis, autoimmune myocarditis, autoimmune vasculitis,dermatomyositis, extrinsic and intrinsic reactive airways disease(asthma), myasthenia gravis, autoimmune ovarian failure, perniciousanemia, Addison's disease, autoimmune hypoparathyroidism or othersyndromes of an inappropriate cellular immune response. The subject canalso have or be at risk of developing another condition related toundesirable memory T cell proliferation.

Selective CDK4/6 inhibitors can also suppress germinal center formation,a process involved in the generation of memory B cells. Thus, in someembodiments, the presently disclosed subject matter provides a method ofreducing or inhibiting B cell progenitor proliferation in a subject inneed of treatment thereof, the method comprising administering to thesubject a pharmaceutically effective amount of a compound thatselectively inhibits CDK4/6. In some embodiments, the subject can haveor be at risk of developing an autoimmune or allergic disease or anothercondition related to undesirable B cell proliferation. In someembodiments, the autoimmune or allergic disease, can be for example,such as, but not limited to, SLE, RA, scleroderma, hemolytic anemia,ITP, aquired inhibitors in hemophilia, TTP, Goodpasture's syndrome, coldand warm agglutin diseases, cryoglobulinemia, or a syndrome ofinappropriate antibody production.

In some embodiments, the presently disclosed subject matter provides amethod of mitigating an autoimmune or allergic disease in a subject inneed of treatment thereof, the method comprising administering to thesubject a pharmaceutically effective amount of a compound thatselectively inhibits CDK4/6, wherein said compound reduces or inhibitsmemory T cell proliferation, B cell progenitor proliferation, or bothmemory T cell proliferation and B cell progenitor proliferation. In someembodiments, the autoimmune disease is selected from the groupincluding, but not limited to, SLE, RA, autoimmune arthritis,scleroderma, hemolytic anemia, autoimmune aplastic anemia, autoimmunegranulocytopenia, type I diabetes, TTP, psoriasis, inflammatory boweldisease, Crohn's disease, ulcerative colitis, contact dermatitis,polymyalgia rheumatica, uveitis, immune pneumonitis, autoimmunehepatitis, immune nephritis, immune glomerulonephritis, multiplesclerosis, autoimmune neuropathy, vitiligo, discoid lupus, Wegener'sGranulomatosis, Henoch-Schoelein Purpura, sclerosing cholangitis,autoimmune thyroiditis, autoimmune myocarditis, autoimmune vasculitis,dermatomyositis, extrinsic and intrinsic reactive airways disease(asthma), myasthenia gravis, autoimmune ovarian failure, perniciousanemia, Addison's disease, autoimmune hypoparathyroidism, othersyndromes of an inappropriate cellular immune response, Goodpasture'ssyndrome, cold and warm agglutin diseases, cryoglobulinemia, or asyndrome of inappropriate antibody production.

In some embodiments, the selective CDK4/6 inhibitor can be used in amethod of treating cancer characterized by an increased level of CDK2activity or by reduced expression of retinoblastoma tumor suppressorprotein or a retinoblastoma family member protein or proteins (such as,but not limited to p107 and p130), the method comprising administeringto the subject a pharmaceutically effective amount of a compound thatselectively inhibits CDK4 and/or CDK6. In some embodiments, theincreased level of CDK2 activity is associated with MYC protooncogeneamplification or overexpression and/or the overexpression of Cylcin E1,E2, or Cylin A. The selective CDK4/6 inhibitor is not believed to inducepharmacologic quiescence in cancer cells in these types of cancers.However, the presently disclosed subject matter is related to the beliefthat selective CDK4/6 inhibitors can increase the sensitivity of cancercells of certain types of cancers to DNA damaging agents, such aschemotherapeutic compounds and ionizing radiation. Thus, in someembodiments, the use of selective CDK4/6 inhibitors can increase thesensitivity of certain types of cancer cells to damage by DNA damagingagents, such as chemotherapeutic compounds or IR, thereby increasingcancer cell death in comparison to when the DNA damaging agent is usedin the absence of administration of the selective CDK4/6 inhibitor.Thus, in some embodiments, a combination of treatment with a DNAdamaging agent and a CKD4/6 inhibitor compound can provide a greaterreduction in tumor burden than treatment with the DNA damaging agentalone. In some embodiments, administration of the compound thatselectively inhibits CDK4 and/or CDK6 can mitigate hematologic toxicityassociated with exposure to a DNA damaging agent or event, such as achemotherapeutic compound or IR. In some embodiments, the hematologictoxicity is a long-term toxicity, such as, but not limited tomyelodysplasia. The administration of the selective CDK4/6 inhibitorcompound can also protect against other long-term toxicities associatedwith exposure to the DNA damaging agent or event, including bothhematologic and non-hematologic toxicities, such as hematologic andnon-hematologic secondary malignancies.

The compound that selectively inhibits CDK4/6 can be administered at anysuitable time prior to, during, or after exposure of the subject to theDNA damaging agent or event. In some embodiments, the selective CDK4/6inhibitor is administered to the subject between about 24 and about 48hours (e.g., about 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48 hours) after exposureof the subject to the DNA damaging agent or event.

Subjects who have been treated for cancer using radiation orchemotherapy have been found to have a higher risk of developing furthercancers (i.e., secondary malignancies, such as cancers that have spreadfrom the original location or new cancers), even when the originalcancer treatment successfully eliminates or otherwise treats (e.g., bythe reduction of tumor burden) the original (i.e., primary) cancer. Thesecondary malignancy can be, for example, leukemia, or anotherhematologic or non-hematologic cancer. These secondary malignancies cansometimes occur several years (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15,20, 25, 30 or more years) after the original cancer has been treated andcan be related to long-term toxicities of the original cancer treatment.

In some embodiments, the presently disclosed subject matter provides amethod of mitigating chemotherapy-induced or radiation-induced secondarymalignancies of hematological or non-hematological origin in a subject.In some embodiments, the method can comprise administering to thesubject a pharmacologically effective amount of a compound thatselectively inhibits CDK4/6. In some embodiments, the compound thatselectively inhibits CDK4 and/or CDK6 is administered to the subjectprior to or during the same time period that the subject is undergoingchemotherapy or radiation-based therapy to treat a primary malignancy.

III. ACTIVE COMPOUNDS, SALTS AND FORMULATIONS

As used herein, the term “active compound” refers to a selective CDK 4/6inhibitor compound, or a prodrug (such as but not limited to variousesters and other derivatives that can form the selective CDK4/6inhibitor in vitro or in vivo), solvate (such as but not limited to ahydrate) and/or pharmaceutically acceptable salt thereof. The activecompound can be administered to the subject through any suitableapproach. The amount and timing of active compound administered can, ofcourse, be dependent on the subject being treated, on the dosage of DNAdamaging agent to which the subject has been, is being, or isanticipated of being exposed to, on the manner of administration, on thepharmacokinetic properties of the active compound, and on the judgmentof the prescribing physician. Thus, because of subject to subjectvariability, the dosages given below are a guideline and the physiciancan titrate doses of the compound to achieve the treatment that thephysician considers appropriate for the subject. In considering thedegree of treatment desired, the physician can balance a variety offactors such as age and weight of the subject, presence of preexistingdisease, as well as presence of other diseases. Pharmaceuticalformulations can be prepared for any desired route of administration,including but not limited to oral, intravenous, or aerosoladministration, as discussed in greater detail below.

The therapeutically effective dosage of any specific active compound,the use of which is within the scope of embodiments described herein,can vary somewhat from compound to compound, and subject to subject, andcan depend upon the condition of the subject and the route of delivery.As a general proposition, a dosage from about 0.1 to about 200 mg/kg canhave therapeutic efficacy, with all weights being calculated based uponthe weight of the active compound, including the cases where a salt isemployed. In some embodiments, the dosage can be the amount of compoundneeded to provide a serum concentration of the active compound of up tobetween about 1-5 μM or higher. Toxicity concerns at the higher levelcan restrict intravenous dosages to a lower level, such as up to about10 mg/kg, with all weights being calculated based on the weight of theactive base, including the cases where a salt is employed. A dosage fromabout 10 mg/kg to about 50 mg/kg can be employed for oraladministration. Typically, a dosage from about 0.5 mg/kg to 5 mg/kg canbe employed for intramuscular injection. In some embodiments, dosagescan be from about 1 μmol/kg to about 50 μmol/kg, or, optionally, betweenabout 22 μmol/kg and about 33 μmol/kg of the compound for intravenous ororal administration.

In accordance with the presently disclosed methods, pharmaceuticallyactive compounds as described herein can be administered orally as asolid or as a liquid, or can be administered intramuscularly,intravenously or by inhalation as a solution, suspension, or emulsion.In some embodiments, the compounds or salts also can be administered byinhalation, intravenously, or intramuscularly as a liposomal suspension.When administered through inhalation the active compound or salt can bein the form of a plurality of solid particles or droplets having aparticle size from about 0.5 to about 5 microns, and optionally fromabout 1 to about 2 microns.

The pharmaceutical formulations can comprise an active compounddescribed herein or a pharmaceutically acceptable salt thereof, in anypharmaceutically acceptable carrier. If a solution is desired, water isthe carrier of choice with respect to water-soluble compounds or salts.With respect to the water-soluble compounds or salts, an organicvehicle, such as glycerol, propylene glycol, polyethylene glycol, ormixtures thereof, can be suitable. In the latter instance, the organicvehicle can contain a substantial amount of water. The solution ineither instance can then be sterilized in a suitable manner known tothose in the art, and typically by filtration through a 0.22-micronfilter. Subsequent to sterilization, the solution can be dispensed intoappropriate receptacles, such as depyrogenated glass vials. Thedispensing is optionally done by an aseptic method. Sterilized closurescan then be placed on the vials and, if desired, the vial contents canbe lyophilized.

In addition to the active compounds or their salts, the pharmaceuticalformulations can contain other additives, such as pH-adjustingadditives. In particular, useful pH-adjusting agents include acids, suchas hydrochloric acid, bases or buffers, such as sodium lactate, sodiumacetate, sodium phosphate, sodium citrate, sodium borate, or sodiumgluconate. Further, the formulations can contain antimicrobialpreservatives. Useful antimicrobial preservatives include methylparaben,propylparaben, and benzyl alcohol. An antimicrobial preservative istypically employed when the formulation is placed in a vial designed formulti-dose use. The pharmaceutical formulations described herein can belyophilized using techniques well known in the art.

For oral administration a pharmaceutical composition can take the formof solutions, suspensions, tablets, pills, capsules, powders, and thelike. Tablets containing various excipients such as sodium citrate,calcium carbonate and calcium phosphate are employed along with variousdisintegrants such as starch (e.g., potato or tapioca starch) andcertain complex silicates, together with binding agents such aspolyvinylpyrrolidone, sucrose, gelatin and acacia. Additionally,lubricating agents such as magnesium stearate, sodium lauryl sulfate andtalc are often very useful for tabletting purposes. Solid compositionsof a similar type are also employed as fillers in soft and hard-filledgelatin capsules. Materials in this connection also include lactose ormilk sugar as well as high molecular weight polyethylene glycols. Whenaqueous suspensions and/or elixirs are desired for oral administration,the compounds of the presently disclosed subject matter can be combinedwith various sweetening agents, flavoring agents, coloring agents,emulsifying agents and/or suspending agents, as well as such diluents aswater, ethanol, propylene glycol, glycerin and various like combinationsthereof.

In yet another embodiment of the subject matter described herein, thereis provided an injectable, stable, sterile formulation comprising anactive compound as described herein, or a salt thereof, in a unit dosageform in a sealed container. The compound or salt is provided in the formof a lyophilizate, which is capable of being reconstituted with asuitable pharmaceutically acceptable carrier to form a liquidformulation suitable for injection thereof into a subject. When thecompound or salt is substantially water-insoluble, a sufficient amountof emulsifying agent, which is physiologically acceptable, can beemployed in sufficient quantity to emulsify the compound or salt in anaqueous carrier. Particularly useful emulsifying agents includephosphatidyl cholines and lecithin.

Additional embodiments provided herein include liposomal formulations ofthe active compounds disclosed herein. The technology for formingliposomal suspensions is well known in the art. When the compound is anaqueous-soluble salt, using conventional liposome technology, the samecan be incorporated into lipid vesicles. In such an instance, due to thewater solubility of the active compound, the active compound can besubstantially entrained within the hydrophilic center or core of theliposomes. The lipid layer employed can be of any conventionalcomposition and can either contain cholesterol or can becholesterol-free. When the active compound of interest iswater-insoluble, again employing conventional liposome formationtechnology, the salt can be substantially entrained within thehydrophobic lipid bilayer that forms the structure of the liposome. Ineither instance, the liposomes that are produced can be reduced in size,as through the use of standard sonication and homogenization techniques.The liposomal formulations comprising the active compounds disclosedherein can be lyophilized to produce a lyophilizate, which can bereconstituted with a pharmaceutically acceptable carrier, such as water,to regenerate a liposomal suspension.

Pharmaceutical formulations also are provided which are suitable foradministration as an aerosol by inhalation. These formulations comprisea solution or suspension of a desired compound described herein or asalt thereof, or a plurality of solid particles of the compound or salt.The desired formulation can be placed in a small chamber and nebulized.Nebulization can be accomplished by compressed air or by ultrasonicenergy to form a plurality of liquid droplets or solid particlescomprising the compounds or salts. The liquid droplets or solidparticles should have a particle size in the range of about 0.5 to about10 microns, and optionally from about 0.5 to about 5 microns. The solidparticles can be obtained by processing the solid compound or a saltthereof, in any appropriate manner known in the art, such as bymicronization. Optionally, the size of the solid particles or dropletscan be from about 1 to about 2 microns. In this respect, commercialnebulizers are available to achieve this purpose. The compounds can beadministered via an aerosol suspension of respirable particles in amanner set forth in U.S. Pat. No. 5,628,984, the disclosure of which isincorporated herein by reference in its entirety.

When the pharmaceutical formulation suitable for administration as anaerosol is in the form of a liquid, the formulation can comprise awater-soluble active compound in a carrier that comprises water. Asurfactant can be present, which lowers the surface tension of theformulation sufficiently to result in the formation of droplets withinthe desired size range when subjected to nebulization.

As indicated, both water-soluble and water-insoluble active compoundsare provided. As used herein, the term “water-soluble” is meant todefine any composition that is soluble in water in an amount of about 50mg/mL, or greater. Also, as used herein, the term “water-insoluble” ismeant to define any composition that has a solubility in water of lessthan about 20 mg/mL. In some embodiments, water-soluble compounds orsalts can be desirable whereas in other embodiments water-insolublecompounds or salts likewise can be desirable.

The term “pharmaceutically acceptable salts” as used herein refers tothose salts which are, within the scope of sound medical judgment,suitable for use in contact with subjects (e.g., human subjects) withoutundue toxicity, irritation, allergic response, and the like,commensurate with a reasonable benefit/risk ratio, and effective fortheir intended use, as well as the zwitterionic forms, where possible,of the compounds of the presently disclosed subject matter.

Thus, the term “salts” refers to the relatively non-toxic, inorganic andorganic acid addition salts of compounds of the presently disclosedsubject matter. These salts can be prepared in situ during the finalisolation and purification of the compounds or by separately reactingthe purified compound in its free base form with a suitable organic orinorganic acid and isolating the salt thus formed. In so far as thecompounds of the presently disclosed subject matter are basic compounds,they are all capable of forming a wide variety of different salts withvarious inorganic and organic acids. Although such salts must bepharmaceutically acceptable for administration to animals, it is oftendesirable in practice to initially isolate the base compound from thereaction mixture as a pharmaceutically unacceptable salt and then simplyconvert to the free base compound by treatment with an alkaline reagentand thereafter convert the free base to a pharmaceutically acceptableacid addition salt. The acid addition salts of the basic compounds areprepared by contacting the free base form with a sufficient amount ofthe desired acid to produce the salt in the conventional manner. Thefree base form can be regenerated by contacting the salt form with abase and isolating the free base in the conventional manner. The freebase forms differ from their respective salt forms somewhat in certainphysical properties such as solubility in polar solvents, but otherwisethe salts are equivalent to their respective free base for purposes ofthe presently disclosed subject matter.

Pharmaceutically acceptable base addition salts are formed with metalsor amines, such as alkali and alkaline earth metal hydroxides, or oforganic amines. Examples of metals used as cations, include, but are notlimited to, sodium, potassium, magnesium, calcium, and the like.Examples of suitable amines include, but are not limited to,N,N′-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine,ethylenediamine, N-methylglucamine, and procaine.

The base addition salts of acidic compounds are prepared by contactingthe free acid form with a sufficient amount of the desired base toproduce the salt in the conventional manner. The free acid form can beregenerated by contacting the salt form with an acid and isolating thefree acid in a conventional manner. The free acid forms differ fromtheir respective salt forms somewhat in certain physical properties suchas solubility in polar solvents, but otherwise the salts are equivalentto their respective free acid for purposes of the presently disclosedsubject matter.

Salts can be prepared from inorganic acids sulfate, pyrosulfate,bisulfate, sulfite, bisulfite, nitrate, phosphate,monohydrogenphosphate, dihydrogenphosphate, metaphosphate,pyrophosphate, chloride, bromide, iodide such as hydrochloric, nitric,phosphoric, sulfuric, hydrobromic, hydriodic, phosphorus, and the like.Representative salts include the hydrobromide, hydrochloride, sulfate,bisulfate, nitrate, acetate, oxalate, valerate, oleate, palmitate,stearate, laurate, borate, benzoate, lactate, phosphate, tosylate,citrate, maleate, fumarate, succinate, tartrate, naphthylate mesylate,glucoheptonate, lactobionate, laurylsulphonate and isethionate salts,and the like. Salts can also be prepared from organic acids, such asaliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoicacids, hydroxy alkanoic acids, alkanedioic acids, aromatic acids,aliphatic and aromatic sulfonic acids, etc. and the like. Representativesalts include acetate, propionate, caprylate, isobutyrate, oxalate,malonate, succinate, suberate, sebacate, fumarate, maleate, mandelate,benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, phthalate,benzenesulfonate, toluenesulfonate, phenylacetate, citrate, lactate,maleate, tartrate, methanesulfonate, and the like. Pharmaceuticallyacceptable salts can include cations based on the alkali and alkalineearth metals, such as sodium, lithium, potassium, calcium, magnesium andthe like, as well as non-toxic ammonium, quaternary ammonium, and aminecations including, but not limited to, ammonium, tetramethylammonium,tetraethylammonium, methylamine, dimethylamine, trimethylamine,triethylamine, ethylamine, and the like. Also contemplated are the saltsof amino acids such as arginate, gluconate, galacturonate, and the like.See, for example, Berge et al., J. Pharm. Sci., 1977, 66, 1-19, which isincorporated herein by reference.

EXAMPLES

The following Examples provide illustrative embodiments and are notintended to limit the scope of the presently disclosed subject matter inany way. In light of the present disclosure and the general level ofskill in the art, those of skill can appreciate that the followingExamples are intended to be exemplary only and that numerous changes,modifications, and alterations can be employed without departing fromthe scope of the presently disclosed subject matter.

The practice of the presently disclosed subject matter can employ,unless otherwise indicated, conventional methods of protein chemistry,biochemistry, recombinant DNA techniques and pharmacology, within theskill of the art. Such techniques are explained fully in the literature.See, e.g., T. E. Creighton, Proteins: Structures and MolecularProperties (W.H. Freeman and Company, 1993); A. L. Lehninger,Biochemistry (Worth Publishers, Inc., current edition); Sambrook et al.,Molecular Cloning: A Laboratory Manual (2^(nd) Edition, 1989); Methodsin Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.);Remington's Pharmaceutical Sciences, 18^(th) Edition (Easton, Pa.: MackPublishing Company, 1990); Carey and Sundberg, Advanced OrganicChemistry 3^(rd) Ed. (Plenum Press) Vols A and B (1992).

Example 1 Synthesis of PD

Scheme 1: Synthesis of PD.

PD was synthesized as shown above in Scheme 1. Reactions shown in Scheme1 generally followed previously reported procedures (see VandelWel etal., J. Med. Chem., 48, 2371-2387 (2005); and Toogood et al., J. Med.Chem., 48, 2388-2406 (2005)), with the exceptions of the reactionconverting compound D to compound E and the reaction converting compoundF to compound G.

Conversion of Compound D to Compound E:

Compound D (40 g, 169 mmol) was dissolved in anhydrous THF (800 mL)under nitrogen and the solution was cooled in ice bath, to which MeMgBrwas added slowly (160 mL, 480 mmol, 3 M in ether) and stirred for 1 h.The reaction was quenched with saturated aqueous NH₄Cl the partitionedbetween water and EtOAc. The organic layer was separated and the aqueouslayer was extracted with EtOAc. The combined organic were washed withbrine and dried over MgSO₄. Concentration gave an intermediate productas an oil (41.9 g, 98%).

The above intermediate (40 g, 158 mmol) was dissolved in dry CHCl₃ (700mL). MnO₂(96 g, 1.11 mol) was added and the mixture was heated to refluxwith stirring for 18 h and another MnO₂ (34 g, 395 mmol) was added andcontinue to reflux for 4 h. The solid was filtrated through a Celite padand washed with CHCl₃. The filtrate was concentrated to give a yellowsolid compound E (35 g, 88%), Mp: 75.8-76.6° C.

Conversion of Compound F to Compound G:

Compound F (5 g, 18.2 mmol) was dissolved in anhydrous DMF (150 mL) andNBS (11.3 g, 63.6 mmol) was added. The reaction mixture was stirred atr.t. for 3.5 h and then poured into H₂O(500 mL), the precipitate wasfiltered and washed with H₂O. The solid recrystallized from EtOH to givecompound G as a white solid (5.42 g, 80.7%), mp: 210.6-211.3° C.

Characterization Data for PD:

LC-MS: 448.5 (ESI, M+H). Purity: ˜99%

¹H NMR (300 MHz, D₂O): 9.00 (s, 1H), 8.12 (dd, J=9.3 Hz, 2.1 Hz, 1H),7.81 (d, J=2.4 Hz, 1H), 7.46 (d, J=9.6 Hz, 1H), 5.80-5.74 (m, 1H),3.57-3.48 (m, 8H), 2.48 (s, 3H), 2.37 (s, 3H), 2.13-1.94 (m, 6H),1.73-1.71 (m, 2H).

¹³C NMR (75 MHz, D₂O): 203.6, 159.0, 153.5, 153.3, 152.2, 139.9, 139.4,139.2, 133.1, 129.0, 118.7, 113.8, 107.4, 51.8, 42.2, 40.0, 28.0, 25.2,22.6, 10.8.

Example 2 General Methods for In Vitro and In Vivo Studies

Compounds: PD0332991 was Synthesized as Described in Example 1.

Cells, cell cycle analysis, γH2AX by flow cytometry, cell proliferationassays and cellular toxicity: Primary normal human renal proximalepithelial cells (American Type Culture Collection (ATCC), Manassas,Va., United States of America) were cultured in renal epithelial cellbasal media supplemented with renal epithelial cell growth kit accordingto the manufacturer's recommendations. Cell cycle analysis was performedusing BrdU (BD Biosciences Pharmingen, San Diego, Calif.) or EdU(Invitrogen Corporation, Carlsbad, Calif., United States of America) andpropidium iodide following the manufacturer's protocols. For the γH2AXassay, cells were fixed, permeabilized, and stained with anti-γH2AX asper γH2AX Flow Kit (Millipore, Billerica, Mass., United States ofAmerica). γH2AX levels were assessed by flow cytometry. Cellproliferation was assessed by seeding 1×10³ cells per well in a 96-welltissue culture plate in 100 μL of growth medium. Cells were treated asindicated with Cdk4/6 inhibitors and etoposide. Following treatment,cells were allowed to recover for 7 days in normal growth medium. At theend of the recovery period, cell number was quantified usingCellTiter-Glo® (Promega, Fitchburg, Wis., United States of America) orWST-1 reagent (TaKaRa Bio USA, Madison, Wis., United States of America).Cellular cytotoxicity was assessed using the TOXILIGHT™ Bioassay kit(Lonza, Basel, Switzerland) which measures cytolysis by quantifying therelease of Adenylate Kinase into the culture media. Briefly, 20 μL wasaspirated from each well of 96 well plates of cells treated with varyingconcentrations of PD0332991. 100 μL of TOXILIGHT™ reagent is added andincubated for 5 minutes and read in a luminometer at 1 second/well.

Animals: All animal experiments were performed in accord with the UNCInstitutional Animal Care and Use Committee. Young adult C57Bl/6 and FVBmice were irradiated using a 137Cs AECL GammaCell 40 Irradiator (AtomicEnergy of Canada Ltd., Mississauga, Ontario, Canada). Mice analyzed wereyoung adult (8-12 weeks of age) virgin female C57Bl/6 or FVB purchasedfrom Jackson Labs (Bar Harbor, Me., United States of America), unlessotherwise specified.

The C3-TAg mice are a model of basal-like breast cancer. The C3-TAg micecontain a recombinant gene expressing the simian virus 40 early-regiontransforming sequence (SV40 large T antigen), which has been shown toinactivate both p53 and Rb. The MMTV-c-neu model expresses c-neu (themouse ortholog of human HER2) driven by the mouse mammary tumor virus(MMTV) promoter and is a model of HER2+ breast cancer. When tumors werenoted to be ˜0.2 cm² in size, animals were treated as described andtumor response assessed by daily caliper measurements.

Drug preparation and dosing: PD0332991 was dissolved in sodium lactatebuffer (pH 4.0) to a final concentration of 15 mg/ml. Mice were treatedwith a 150 mg/kg dose of PD0332991. 2BrIC (also referred to herein asL4D) was solubilized in DMSO and added to cells where finalconcentration of DMSO<0.1%.

Analysis of BrdU Incorporation: For kidney proliferation experiments,mice were treated with a single dose of PD0332991 (150 mg/kg oralgavage) or vehicle control followed by cisplatin (10 mg/kg IP).Proliferation was assessed by using BrdU (1 mg IP injection) every 6hours for 24 hours prior to sacrifice or 100 μg of EdU (0.1 mg IP) every24 hours for 3 days prior to sacrifice.

Analysis of EdU incorporation by flow cytometry: Kidneys were harvestedfrom mice and single cells were isolated using a gentleMACS™ tissuedissociator (Miltinyi Biotec, Bergisch Gladbach, Germany). Briefly,kidneys were cut into small pieces and placed in 10 ml collagenase (1mg/ml) in a gentleMACS™ C tube (Miltinyi Biotec, Bergisch Gladbach,Germany). Tissue was dissociated following the manufacturer'srecommendations (Miltinyi Biotec, Bergisch Gladbach, Germany). Cellswere then incubated for 5 minutes in ACK buffer to lyse red blood cells,filtered and pelleted. Cells were resuspended in 4% paraformaldehyde andstored overnight at 4° C. For quantification of EdU incorporation, thecells were fixed, permeablized, and stained with an APC EdU Flow Kitaccording to the manufacturer's instruction (Invitrogen Corporation,Carlsbad, Calif., United States of America). Flow cytometric analysiswas performed using a CyAn ADP (Dako, Glostrup, Denmark). For eachsample, a minimum of 500,000 cells was analyzed and the data wereanalyzed using FlowJo software (Tree Star, Inc., Ashland, Oreg., UnitedStates of America).

Myelosuppression Assay: Weekly Complete Blood Counts: In theradioprotection experiments, mice were treated with a PD0332991 150mg/kg by oral gavage or vehicle control one hour before exposure toradiation (6.5 Gy). Erythropoietin (4000 units/day) was given beginningon day 3 following exposure to radiation and continued for threeconsecutive days.

Baseline complete blood cell (CBC) anaylsis was performed on a subset ofmice prior to drug administration. Following drug administration(chemotherapy/radiation+/−CDK4/6 inhibitor/erythropoietin or control),CBC analysis was performed on day 10 and 17 following treatment. 40 μlof blood was collected by tail vein nick in BD Microtainer tubes withK2E (K₂EDTA). Blood was analyzed using a Hemavet CBC-Diff VeterinaryHematology System (Drew Scientific Inc., Dallas, Tex., United States ofAmerica). CBC analysis included measurement of white blood cells,lymphocytes, granulocytes, monocytes, hematocrit, red blood cells,hemoglobin, platelets, and other common hematological parameters.

Statistical Analysis: Unless otherwise noted, comparisons are made withone-way ANOVA with Bonferroni correction for multiple comparisons whereappropriate. Error bars are +/−standard error of the mean (SEM) orstandard deviation as indicated.

Example 3 Augmentation of Growth Factor Efficacy by CDK4/6 Inhibition

Cohorts of FVB wild type mice were given placebo or CDK4/6 inhibitor(PD0332991, 150 mg/kg oral gavage) just prior to receiving a sub-lethaldose of irradiation (6.5 Gcy). Three doses of normal saline (control) orerythropoietin (EPO) 100 units were administered by subcutaneousinjection at times 72, 96, and 120 hours post-irradiation. In total,there were four treatment cohorts (PD0332991+Saline, PD0332991+EPO,Saline+EPO, Saline+Saline). The sample size for each cohort was:Control=7; EPO=8; PD0332991=8, PD/EPO=6. Serial blood draws wereperformed at baseline, 10 days post irradiation, and 17 days postirradiation. Complete blood counts (CBCs) were assessed to determine thenumber of red blood cells, various leukocytes subpopulations andplatelets.

EPO alone or in combination with PD0332991 had no effect on platelets(FIG. 1) or other non-erythroid cell lineages, whereas both treatmentcohorts that received PD0332991 showed improved platelet counts (FIG. 1)as well as other non-erythroid cell lineages. EPO alone was not able toimprove erythroid cell lineage. Without being bound to any one theory,this is believed to be because EPO treatment stimulated erythroidprogenitors harboring DNA damage to enter the cell cycle resulting insubsequent apoptosis. However, treatment of mice with PD0332991 incombination with EPO showed marked improvement in erythroid function asshown by improved RBC, Hb, and HCT measurements. Again without beingbound to any one theory, it is believed that PD0332991 allows erythroidprogenitors to repair DNA damage from radiation and then subsequent EPOtreatment is believed to stimulate the progenitors to expedite erythroidreplacement. In conclusion, CDK4/6 inhibitors appear to enhance theefficacy of growth factors to rescue and support the varioushematopoietic populations following exposure to DNA damaging agents suchas radiation or chemotherapy. Thus, for example, as part ofchemotherapy-based cancer treatment regimes, CDK4/6 inhibition aroundthe time of DNA damage can be used to enhance growth factor support ofbone marrow suppression by allowing bone marrow stem and progenitors torepair DNA damage before growth factor administration has begun.Further, CDK4/6 inhibition will mitigate long term (e.g., 3 or moreyears post chemotherapy) bone marrow toxicities (for example,myelodysplasia) related to the use of growth factors in cancer patientswho survive the disease.

CDK4/6 inhibition around the time of DNA damaging exposure can augmentthe efficacy of growth factors such as (but not limited to) G-CSF andderivatives (e.g. pegylated G-CSF), GM-CSF and derivatives,thrombopoietin and derivatives, erythropoietin and derivatives (e.g.pegylated erythropoietin), IL12, steel factor, Keratinocyte growthfactors. These agents, especially O—CSF, GM-CSF and erythropoietin andderivatives, are clinically used to reduce the toxicity of chemotherapyand radiation in the care of cancer patients. Pharmacologic quiescenceinduction through CDK4/6 inhibition around the time of DNA damagingexposure can augment the efficacy of these agents at a later time point(e.g., growth factors administration is usually begun 24-72 after theDNA damaging therapeutic).

Example 4 Protection of Non-Hematologic Tissues and Cells by CDK4/6Inhibition

Use of a potent and selective CDK4/6 inhibitor, such as PD0332991,induces a G1 arrest in normal human primary renal proximal tubuleepithelial cells. See FIGS. 2A and 2B. A dose dependent increase in theG0/G1 fraction of the cell cycle was observed with a consummate decreasein both G2/M and S-phase fractions. In doing so, the cells enterpharmacologic quiescence and are held in this state until they arereleased from this arrest.

Normal human primary renal proximal tubule epithelial cells were platedand exposed 24 hours later to PD0332991 at concentrations of 0, 10 nM,30 nM, 100 nM, 300 nM or 1 uM. Sixteen hours post treatment; cells wereharvested by standard methods, fixed in ice-cold methanol until time forDNA staining. Samples were processed and the DNA was stained withpropidium iodide (PI) solution and analyzed by flow cytometry. FCS filesfrom flow cytometer were further analyzed using cell cycle analysissoftware Mod-Fit™ from Verity (Verity Software House, Topsham, Me.,United States of America), where cell cycle fractions were calculated asa percentage of the whole population.

Inhibition of CDK4/6 blocks the proliferation of normal human primaryrenal proximal tubule epithelial cells. These cells were seeded at anappropriate density in 96 well plates and incubated for 24 hours at 37°C. in a humidified incubator at 5% CO₂. Cells were then exposed to apotent and selective Cdk4/6 inhibitor, in this case PD0332991, across abroad dose range 24 hours later. The dose range explored is 0, 10 nM, 30nM, 100 nM, 300 nM, 1 μM or 3 μM PD0332991. Seventy-two hours postexposure, the CDK4/6 inhibited cells were treated with CellTiter-Glo®(Promega, Madison, Wis., United States of America) using manufacturer'sspecifications. The plate was read in luminometer at 1 second/well.Results were placed in Microsoft Excel and analyzed. In FIG. 3, a cleardose dependent inhibition of cell proliferation is obtained in thepresence of this inhibitor when compared to DMSO control by 72 hourspost treatment. This result, in conjunction with FIGS. 2A and 2Bdemonstrates that Cdk4/6 dependent non-hematologic cells can enterpharmacologic quiescence and are thusly inhibited from proliferating.

CDK4/6 inhibition abrogates etoposide-induced DNA damage in normal humanprimary renal proximal tubule epithelial cells. In cell cultures exposedto DNA damaging small molecules or ionizing radiation, double-strandedDNA breaks are generated rapidly which will lead to the phosphorylationof H2AX. Phosphorylation of H2AX corresponds with double stranded DNAbreaks. In FIG. 4, normal human primary renal proximal tubule epithelialcells were plated and treated them 24 hours later with 0, 100 nM, 300 nMor 1 μM PD0332991. Sixteen hours later, these samples were exposed to2.5 μM etoposide for eight hours. Samples were then harvested, fixed andstained for γH2AX using Millipore Corporation H2AXx PhosphorylationAssay Kit for Flow Cytometry (Millipore, Billerica, Mass., United Statesof America). Samples were run on our flow cytometer and resultsprocessed through FlowJo Flow Cytometry Analytical Software (Treestar,Inc., Ashland, Oreg., United States of America). These resultsdemonstrate that pharmacologic quiescence provides protection ofchemotherapeutically induced DNA damage through pharmacoquiesence in adose dependent manner.

CDK4/6 inhibition protects normal human primary renal proximal tubuleepithelial cells from etoposide-induced cell death. In FIG. 5, it isdemonstrated that the use of a selective and potent CDK4/6 inhibitor innon-hematologic cells dependent of CDK4/6 can provide protection fromDNA damaging agents, such as, but not limited to, etoposide. Normalhuman primary renal proximal tubule epithelial cells were plated andtreated with increasing doses of the CDK4/6 inhibitor PD0332991 24 hoursafter seeding. Sixteen hours after treatment, these cells were dosedwith 2.5 μM etoposide for 8 hours. Media was removed and replace withfresh media. Cells were maintained in culture for 7 days at which theywere evaluated with CellTiter-Glo® (Promega, Madison, Wis., UnitedStates of America) using manufacturer's specifications for effects oncell proliferation. The plate was read in luminometer at 1 second/well.Results were placed in Microsoft Excel and analyzed. Cells treated withincreasing doses on PD0332991 exhibit in a dose dependent mannerprotection from etoposide induced cell death.

The kidney is relatively quiescent until challenged by a renal insult.Therefore, to determine whether renal cell proliferation was dependenton CDK4/6 activity in vivo, renal cell proliferation was stimulated bytreating female FVB wt mice with cisplatin, a known nephrotoxicchemotherapeutic agent. At time 0 hr, mice were started on chowdelivering PD0332991 100 mg/kg per day or standard chow with no drug. At24 hours mice received a single dose of cisplatin 15 mg/kg by IPinjection and an IP injection of 100 mcg of EdU. At 48 hours all micereceived a second dose of 100 mcg EdU by IP injection. After 72 hoursmice were euthanized and kidneys were harvested. Single cell suspensionsof renal cells were made by gently grinding the kidneys using thegentleMACS™ tissue dissociator (Miltinyi Biotec, Bergisch Gladbach,Germany). Single cell suspensions were then used to measure EdUincorporation flow cytometric analysis. Mice treated with cisplatin andvehicle control showed approximately 17% of cells labeled with EdU,whereas mice treated with cisplatin and PD0332991 only had approximately2% of cells stained positive for EdU incorporation. See FIG. 6. Thus,CDK4/6 inhibition resulted in an 88% reduction in cell proliferation,further confirming the in vitro analysis that renal cell proliferationis dependent on CDK4/6 activity.

To determine if CDK4/6 inhibition around the time of DNA damage wouldprotect renal function, mice were treated with cisplatin, a knowncausative agent of renal tubular damage in humans. Mice were treatedwith PD0332991 150 mg/kg or vehicle control by oral gavage and thenreceived a single dose of cisplatin 15 mg/kg by IP injection. 72 hourspost treatment mice were euthanized and blood was collected by cardiacpuncture for BUN (blood urea nitrogen) and serum creatinine (SrCr)analysis. Serum BUN and SrCr are common markers of renal function andserum levels quickly elevated when kidney function has been acutelycompromised. FIG. 7 shown a dramatic increase in BUN and SrCr followingcisplatin administration and a single dose of PD0332991 co-administeredwith the cisplatin was able to abrogate the cisplatin-inducednephrotoxicity.

CDK4/6 appears to play a role in cell proliferation of certainnon-hematological tissues, such as the kidney. Thus, CDK4/6 inhibitorscan be used to protect non-hematological tissues, such as, but notlimited to, kidney, gut, heart, liver, brain, thyroid, skin, intestinalmucosa, auditory system, lung, bladder, ovaries, uterus, testicles,adrenals, gallbladder, pancreas and pancreatic islets, stomach, bloodvessels, and bone, from DNA damaging agents such as radiation andchemotherapy.

Example 5 Augmentation of DNA Damaging Agent Efficacy by CDK4/6Inhibition

The proliferative effects of CDK4/6 inhibition on a panel of small celllung cancer (SCLC) cell lines with intact RB (H417) or that wereRB-deficient (H69, H82, H209, H345) was evaluated. Cells were treatedwith DMSO or PD0332991 100 nM for 48 hours and then cell number wasestimated using the WST-1 assay, a measure of cellular respiration. SeeFIG. 8. In the RB-intact SCLC cell line (H417), cell proliferation wasdecreased, whereas in all four of the RB-deficient cell lines, cellproliferation was actually increased by CDK4/6 inhibition.

The effects of CDK4/6 inhibition in the C3-Tag transgenic mouse model ofbasal-like breast cancer were also evaluated. The C3-TAg model containsa recombinant gene expressing the simian virus 40 early-regiontransforming sequence (SV40 large T antigen), which has been shown toinactivate both p53 and RB. Mice were housed up to five per cage with adlibitum access to standard chow and water. Tumor volume was measured bycaliper weekly. Tumor volume was calculated using the following formula:Volume=[(width)²×length]/2. After establishing sufficient tumor volume(50-60 mm³), mice were stratified by tumor size and randomly assigned toeach of the study cohorts (Untreated, PD0332991 100 mg/kg daily instandard chow, chemotherapy plus vehicle control once a week for 3weeks, or chemotherapy and PD0332991 once a week for three weeks). Inthe once a week for 3 week treatment cohorts, chemotherapy wasadministered by IP injection and PD0332991 150 mg/kg or vehicle controlwas administered by oral gavage. The chemotherapy regimen consisted ofcarboplatin 75 mg/kg once a week for three weeks. Treatments wereadministered on days 0, 7 and 14 and tumor volumes were measured weekly,until the mice died or were euthanized due to toxicity or tumor burden.

Daily administration of the CDK4/6 inhibitor, PD0332991, had no effecton tumor growth in the C3-Tag model at day 21 (see FIG. 9), whereasco-administration of PD0332991 150 mg/kg with carboplatin 75 mg/kg oncea week for 3 weeks resulted in enhanced tumor response in the C3-Tagmice (FIG. 9). In addition, long-term follow-up of the C3-Tag miceshowed that tumor progression was delayed in the PD0332991/Carboplatincohort compared to the Mock Gavage/Carboplatin cohort (FIG. 10).Together these data suggest that, in the treatment of tumors with severederangements of the cell cycle, CDK4/6 inhibition can enhance theefficacy of chemotherapy.

Accordingly, it appears that CDK4/6 inhibition can augment the efficacyof DNA damaging agents in the treatment of certain cancers with severederangements of the cell cycle, for example, cancers characterized byvery high levels of CDK2 activity (e.g. as a result of amplification ofthe MYC proto-oncogene) or loss of the RB tumor suppressor protein. Insuch tumors, CDK4/6 inhibitors do not induce pharmacological quiescencein the tumor cells, but rather increase the sensitivity of the cancer toDNA damaging agents, thereby increasing tumor kill. CDK4/6 inhibitortreatment simultaneously prevents the host hematologic toxicity of DNAdamaging agents (through the induction of quiescence in certain othercells). This increase in tumor kill of RB-null or MYC amplified cancerscombined with decreased host toxicity means an increase in thetherapeutic window of such tumors, allowing for such tumors to be moreeasily cured with less toxicity to the patient.

A subset of tumor types such as Her2 amplified breast cancers areexpected to be sensitive to CDK4/6 inhibition and thus co-administrationof CDK4/6 inhibitor with chemotherapy is likely to result in tumorprotection. However, most cancers appear to use the proliferativekinases promiscuously (e.g., can use CDK 1/2/4/or 6). Therefore,isolated inhibition of CDK4/6 should not affect tumor growth in themajority of cancers and CDK4/6 inhibition should not negatively impactthe efficacy chemotherapy in these tumor types. In fact, as noted above,CDK4/6 inhibition, with selective small molecule inhibitors, is expectedto increase the efficacy of chemotherapeutic agents in certain tumorsthat are not CDK4/6 dependent. As would be understood by one of skill inthe art, such tumors can be deduced based on tumor type and moleculargenetics, and, for example, can be cancers characterized by one or moreof the group including, but not limited to, increased activity of CDK1or CDK2, loss or absence of retinoblastoma tumor suppressor protein(RB), high levels of MYC expression, increased cyclin E and increasedcyclin A. Such cancers can include, but are not limited to, small celllung cancer, retinoblastoma, HPV positive malignancies like cervicalcancer and certain head and neck cancers, MYC amplified tumors such asBurkitts Lymphoma, and triple negative breast cancer; certain classes ofsarcoma, certain classes of non-small cell lung carcinoma, certainclasses of melanoma, certain classes of pancreatic cancer, certainclasses of leukemia, certain classes of lymphoma, certain classes ofbrain cancer, certain classes of colon cancer, certain classes ofprostate cancer, certain classes of ovarian cancer, certain classes ofuterine cancer, certain classes of thyroid and other endocrine tissuecancers, certain classes of salivary cancers, certain classes of thymiccarcinomas, certain classes of kidney cancers, certain classes ofbladder cancer and certain classes of testicular cancers.

In non-limiting examples, the cancer is selected from a small cell lungcancer, retinoblastoma and triple negative (ER/PR/Her2 negative) or“basal-like” breast cancer. Small cell lung cancer and retinoblastomaalmost always inactivate the retinoblastoma tumor suppressor protein(RB), and therefore does not require CDK4/6 activity to proliferate.Thus, CDK4/6 inhibitor treatment will effect pharmacologic quiescence inthe bone marrow and other normal host cells, but not in the tumor.Triple negative (basal-like) breast cancer is also almost alwaysRB-null. Also, certain virally induced cancers (e.g. cervical cancer andsubsets of Head and Neck cancer) express a viral protein (E7), whichinactivates RB making these tumors functionally RB-null. Some lungcancers are also believed to be caused by HPV. As would be understood byone of skill in the art, cancers that are not expected to be affected byCDK4/6 inhibitors (e.g., those that are RB-null, that express viralprotein E7, or that overexpress MYC) can be determined through methodsincluding, but not limited to, DNA analysis, immunostaining, Westernblot analysis, and gene expression profiling.

Example 6 Blockade of T Cell Proliferation by CDK4/6 Inhibition

Acute, pharmacologic inhibition of CDK4/6 suppresses lymphocyteproliferation with the most pronounced effect on memory T cellhomeostatic proliferation and germinal center formation in mice. Todetermine whether inhibiting CDK4/6 affects memory cell generation andmaintenance, mice were treated with selective CDK4/6 inhibitors, PD0332991 or an unrelated selective CDK4/6 inhibitor, 2BrIC. 2BrIC wassynthesized by OTAVA Chemicals (Kiev, Ukraine) and can be preparedaccording to methods described in Zhu et al., J. Med. Chem. 46,2027-2030 (2003). Acute inhibition of CDK4/6 by PD 0332991 or 2BrICresulted in more significant decrease in homeostatic proliferation ofmemory T cells than naive T cells, as measured by BrdU incorporation andKi67 expression in both human and murine cells. See FIGS. 11, 17, 18 and21. In FIG. 11A, an effect of PD0332991 on in vivo BrdU incorporation ofCD4+ and CD8+ murine Tcells, with greatest effects seen in the CD44+CD25+ memory cells (quantified in FIG. 11B). A similar effect on in vivohomeostatic proliferation was noted in unstimulated splenic T cellsusing Ki67 staining (FIG. 21). Decreased CKD4/6 activity also suppressedgerminal center formation, which is relevant to memory B cellgeneration. See FIG. 11C. These data reveal a role for CDK4/6 in memorycell homeostasis.

Similar results were seen using human lymphocytes. FIG. 12 shows anexperimental scheme to address similar issues in human cells. Humanlymphocytes are sorted to T(CD3+) and B(CD19+) cells and treated invitro with CDK4/6 inhibitor prior to stimulation with PMA and Inomycin(P+I)+OKT3 (Tcells) or IgM (Bcells), with proliferation assessed by BrdUuptake and Ki67y expression, and activation assessed by CD25 expression.FIG. 13 shows that as in murine cells, CDK4/6 inhibitors blockproliferation in response to Tcell receptor (TCR) stimulation (P+I),with a greater effect in CD45RA low memory cells. These data are graphedin FIG. 14. In FIG. 15, the effects of CDK4/6 inhibition on specificTcell fractions is assessed, with and without TCR, showing CDK4/6inhibition has a greater effect on proliferation of memory cellsrelative to naïve cells. A similar effect was seen in CD8+ cells. FIG.16 shows similar data as in FIG. 15, but using Ki67 as a marker ofproliferation instead of BrdU. These effects on proliferation change therelative frequencies of CD4+ (see FIGS. 17A, 17B, and 17C) and CD8+ (seeFIG. 18) cells. CDK4/6 inhibitors decrease the CD4+ effector memory (EM)cell frequency to a greater extent than naïve cells. See FIGS. 17A and17C. A similar result is seen in CD8+ cells. As a result of theseeffects on proliferation, memory/naive ratio decreased by half in bothCD4+ and CD8+ compartments. See FIGS. 17B and 20. These alterations inproliferation are associated with decreased T cell activation asmeasured by CD25 expression. See FIG. 19.

This ability to inhibit T cell proliferation can be of use in thetherapy of autoimmune and allergic diseases. These conditions arepresently treated with a variety of cytotoxic and steroidal agents thathave significant toxicity. The memory T cell compartment has beendifficult to target in order to attenuate anemnestic immune responses,and the use of CDK4/6 inhibitors to reduce proliferation of thisfraction will be particularly useful for therapy of autoimmune andallergic diseases.

Thymocyte differentiation: The effect of CDK4/6 inhibition on thymocytedevelopment was assessed by determining the percentages and absolutenumbers of thymocytes at different stages (Double Negative (DN): CD4−CD8−; Double Positive (DP): CD4+ CD8+; Single Positive (SP): CD4+ orCD8+) by FACS. DN cells are converted to DP cells which are thenconverted to cells singly positive for CD4 or CD8. Transient CDK4/6inhibition produced a pronounced reduction in DP and SP cells, withrelative sparing of DN cells. This result suggests that CDK4/6activation is required during thymopoiesis for the production of newnaïve Tcells. See FIG. 23.

Example 7 Blockade of B Cell Proliferation by CDK4/6 Inhibition

Cohorts of wild type mice were treated with vehicle or a CDK4/6inhibitor as in FIG. 11. Ki67 staining of a germinal center in a lymphnode shows a marked decreased in proliferation with CDK4/6 inhibition. Asimilar result was seen in splenic CD45R+B-cells. See FIG. 21.Unstimulated mice were treated for 24 hours with PD0332991 andhomeostatic B cell proliferation measured by Ki67 staining afterappropriate sorting. Similar results were obtained using BrdUincorporation to measure splenic B cell proliferation. See FIG. 22.Similar experiments were undertaken in human cells with B cell receptorstimulation as described in FIG. 12. FIG. 20 shows that CDK4/6inhibition blocks Bcell stimulation by P+I. These results show thathomeostatic, germinal center and BCR-induced Bcell proliferationrequires CDK4/6 activity in mice and humans.

Example 8 Suppression of Autoimmune Disease Development by CDK4/CDK6Inhibition

Several lines of autoimmune mouse models have been developed, includingNOD mice (spontaneous autoimmune diabetes) and Lyn−/− (lupus likeautoimmune disease). See, e.g., Anderson and Bluestone, Annual Review ofImmunology 23, 447-485 (2005); and Hibbs et al., Cell 83, 301-311(1995)). Cohorts of both young (about 4-6 weeks) and old (>30 weeks)mice are treated with placebo or a CDK4/6 inhibitor for defined periodsof time before being analyzed for autoimmune phenotypes.

It will be understood that various details of the presently disclosedsubject matter can be changed without departing from the scope of thepresently disclosed subject matter. Furthermore, the foregoingdescription is for the purpose of illustration only, and not for thepurpose of limitation.

1. A method of increasing the efficacy of a toxicity reducing agent in a subject in need of treatment thereof, the method comprising: providing a subject that has been exposed to, is being exposed to, or is at risk of being exposed to a DNA damaging agent or event; administering to said subject a toxicity reducing agent; and administering to said subject a pharmaceutically effective amount of a compound that selectively inhibits cyclin dependent kinase 4 (CDK4) and/or cyclin dependent kinase 6 (CDK6).
 2. The method of claim 1, wherein said toxicity reducing agent is a chemotherapy toxicity reducing agent.
 3. The method of claim 1, wherein said toxicity reducing agent is a radiation toxicity reducing agent.
 4. The method of claim 1, wherein said toxicity reducing agent comprises one or more agents selected from the group consisting of a growth factor, a granulocyte colony-stimulating factor (G-CSF), a pegylated G-CSF, granulocyte-macrophage colony stimulating factor (GM-CSF), thrombopoietin, erythropoietin, pegylated erythropoietin, interleukin (IL)-12, steel factor, a keratinocyte growth factor, or derivatives thereof.
 5. The method of claim 1, wherein the compound that selectively inhibits CDK4 and/or CDK6 induces pharmacologic quiescence in one or more cells within the subject.
 6. The method of claim 5, wherein the one or more cells are each selected from the group consisting of a hematologic cell, a hematologic stem cell, and a hematologic precursor cell.
 7. The method of claim 1, wherein the compound that selectively inhibits CDK4 and/or CDK6 is administered to the subject prior to the subject being exposed to the DNA damaging agent or event, at the same time the subject is being exposed to the DNA damaging agent or event, or after exposure of the subject to the DNA damaging agent or event.
 8. The method of claim 1, wherein the compound that selectively inhibits CDK4 and/or CDK6 is administered to the subject between about 24 and about 48 hours after exposure of the subject to the DNA damaging agent or event.
 9. A method of mitigating DNA damage in a non-hematologic cell or tissue in a subject in need of treatment thereof prior to or following exposure of the cell or tissue to a DNA damaging agent or event, the method comprising administering to the subject a pharmaceutically effective amount of a compound that selectively inhibits cyclin dependent kinase 4 (CDK4) and/or cyclin dependent kinase 6 (CDK6).
 10. The method of claim 9, wherein the non-hematologic cell or tissue is comprises a cell or tissue from one of the group consisting of kidney, gut, heart, liver, brain, thyroid, skin, intestinal mucosa, auditory system, lung, bladder, ovaries, uterus, testicles, adrenals, gallbladder, pancreas, pancreatic islets, stomach, blood vessels, bone, and combinations thereof.
 11. A method of reducing or inhibiting memory T cell proliferation in a subject in need of treatment thereof, the method comprising administering to the subject a pharmaceutically effective amount of a compound that selectively inhibits cyclin dependent kinase 4 (CDK4) and/or cyclin dependent kinase 6 (CDK6) to the subject.
 12. The method of claim 11, wherein the subject has or is at risk of developing an autoimmune or allergic disease.
 13. The method of claim 12, wherein the autoimmune or allergic disease is selected from the group consisting of systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), autoimmune arthritis, scleroderma, hemolytic anemia, autoimmune aplastic anemia, autoimmune granulocytopenia, type I diabetes, thrombotic thrombocytopenic purpura (TTP), psoriasis, inflammatory bowel disease, Crohn's disease, ulcerative colitis, contact dermatitis, polymyalgia rheumatica, uveitis, immune pneumonitis, autoimmune hepatitis, immune nephritis, immune glomerulonephritis, multiple sclerosis, autoimmune neuropathy, vitiligo, discoid lupus, Wegener's Granulomatosis, Henoch-Schoelein Purpura, sclerosing cholangitis, autoimmune thyroiditis, autoimmune myocarditis, autoimmune vasculitis, dermatomyositis, extrinsic and intrinsic reactive airways disease (asthma), myasthenia gravis, autoimmune ovarian failure, pernicious anemia, Addison's disease, autoimmune hypoparathyroidism and other syndromes of inappropriate cellular immune response.
 14. A method of reducing or inhibiting B cell progenitor proliferation in a subject in need of treatment thereof, the method comprising administering to the subject a pharmaceutically effective amount of a compound that selectively inhibits cyclin dependent kinase 4 (CDK4) and/or cyclin dependent kinase 6 (CDK6) to the subject.
 15. The method of claim 14, wherein the subject has or is at risk of developing an autoimmune or allergic disease.
 16. The method of claim 15, wherein the autoimmune or allergic disease is selected from the group consisting of systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), scleroderma, hemolytic anemia, idiopathic thrombocytopenic purpura (ITP), acquired inhibitors in hemophilia, thrombotic thrombocytopenic purpura (TTP), Goodpasture's syndrome, cold and warm agglutin diseases, cryoglobulinemia, and syndromes of inappropriate antibody production.
 17. A method for mitigating an autoimmune or allergic disease in a subject in need of treatment thereof, the method comprising administering to the subject a pharmaceutically effective amount of a compound that selectively inhibits cyclin-dependent kinase 4 (CDK4) and/or cyclin dependent kinase 6 (CDK6), wherein said compound reduces or inhibits memory T cell proliferation, B cell progenitor proliferation, or both memory T cell proliferation and B cell progenitor proliferation.
 18. The method of claim 17, wherein the autoimmune or allergic disease is selected from the group consisting of systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), autoimmune arthritis, scleroderma, hemolytic anemia, autoimmune aplastic anemia, autoimmune granulocytopenia, type I diabetes, thrombotic thrombocytopenic purpura (TTP), psoriasis, inflammatory bowel disease, Crohn's disease, ulcerative colitis, contact dermatitis, polymyalgia rheumatica, uveitis, immune pneumonitis, autoimmune hepatitis, immune nephritis, immune glomerulonephritis, multiple sclerosis, autoimmune neuropathy, vitiligo, discoid lupus, Wegener's Granulomatosis, Henoch-Schoelein Purpura, sclerosing cholangitis, autoimmune thyroiditis, autoimmune myocarditis, autoimmune vasculitis, dermatomyositis, extrinsic and intrinsic reactive airways disease (asthma), myasthenia gravis, autoimmune ovarian failure, pernicious anemia, Addison's disease, autoimmune hypoparathyroidism, other syndromes of an inappropriate cellular immune response, Goodpasture's syndrome, cold and warm agglutin diseases, cryoglobulinemia, and syndromes of inappropriate antibody production.
 19. A method of treating cancer in a subject in need of treatment thereof, wherein the cancer is characterized by an increased level of cyclin dependent kinase 2 (CDK2) activity or by reduced expression of retinoblastoma tumor suppressor protein or a retinoblastoma family member protein, the method comprising administering to the subject a pharmaceutically effective amount of a compound that selectively inhibits cyclin dependent kinase 4 (CDK4) and/or cyclin dependent kinase 6 (CDK6).
 20. The method of claim 19, wherein the compound that selectively inhibits CDK4 and/or CDK6 does not induce pharmacologic quiescence in cancer cells.
 21. The method of claim 19, wherein the compound that selectively inhibits CDK4 and/or CDK6 increases the sensitivity of cancer cells to DNA damaging agents.
 22. The method of claim 21, wherein the increase in sensitivity increases cancer cell death.
 23. The method of claim 19, wherein the increased level of CDK2 activity is associated with MYC protooncogene amplification or overexpression.
 24. The method of claim 19, wherein the increased level of CDK2 activity is associated with overexpression of Cyclin E1, Cyclin E2, or Cyclin A.
 25. The method of claim 19, wherein administration of the compound that selectively inhibits CDK4 and/or CDK6 mitigates hematologic toxicity associated with exposure to a DNA damaging agent or event.
 26. The method of claim 25, wherein the compound that selectively inhibits CDK4 and/or CDK6 is administered to the subject prior to the subject being exposed to the DNA damaging agent or event, at the same time the subject is being exposed to the DNA damaging agent or event, or after exposure of the subject to the DNA damaging agent or event
 27. The method of claim 25, wherein the compound that selectively inhibits CDK4 and/or CDK6 is administered to the subject between about 24 and about 48 hours after exposure of the subject to the DNA damaging agent or event.
 28. A method of mitigating chemotherapy-induced or radiotherapy-induced secondary malignancies of hematological or non-hematological origin in a subject, the method comprising administering to the subject a pharmacologically effective amount of a compound that selectively inhibits cyclin dependent kinase 4 (CDK4) and/or cyclin dependent kinase 6 (CDK6).
 29. The method of claim 28, wherein the compound that selectively inhibits CDK4 and/or CDK6 is administered to the subject prior to or during the same time period that the subject is undergoing chemotherapy or radiation-based therapy to treat a primary malignancy. 